The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin-based semen extender as a substitute for egg yolk-based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris-based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post-thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early-apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post-thawed necrotic and late-apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.

Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH-GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte-complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures ‘normal’ intracellular GSH content in COCs and protects DNA integrity of cumulus cells.

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non-domestic animal species. However, problems arise during the development of these embryos, which may be related to species-specific differences in nuclear–cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria-related genes NRF1, MT-CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34–33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45–12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80–87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT-CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.

The objective was to examine the effect of lactation on uterine involution in post-partum dairy cows. Holstein primiparous cows were used (n = 19, mean age: 3.9 ± 0.1 years). At calving, cows were randomly assigned to one of two treatment groups, lactating (n = 11) or non-lactating (i.e. dried off at calving, n = 8). Examination of the reproductive tract was carried out by ultrasonography twice weekly until week 7 post-partum. Blood samples were collected twice weekly for the analysis of progesterone to indicate the resumption of cyclicity and metabolites indicative of energy status. Uterine involution was assessed in terms of size of the uterine horns, uterine body diameter and uterine fluid volume as assessed by the amount of non-echogenic material measured by ultrasound and position of the uterus. Vaginal mucous score was taken on day 28 post-partum for the assessment of uterine inflammation. Resumption of cyclicity (serum progesterone > 1 ng/ml) had occurred in both groups on average by day 21 post-partum. Concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher, whereas concentrations of glucose, insulin and IGF-1 were lower (p < 0.05) in lactating compared to non-lactating cows. Lactating cows had a smaller mean uterine body diameter (p < 0.05) than non-lactating cows from days 28 to 42 post-partum (day 28: 20.2 ± 1.3 vs 24.9 ± 1.5 mm, respectively) and had a lower mean uterine fluid volume up to day 49 (p < 0.05). By day 49, there was no difference in uterine diameter (15.2 ± 1.8 vs 15.2 ± 1.6 mm) or uterine fluid volume (0.11 ± 0.38 vs 0.18 ± 0.46) between lactating and non-lactating cows, respectively. Vaginal mucous score revealed no evidence of uterine inflammation in either group. In conclusion, while lactation induced significant alterations in metabolic status, it did not have a major effect on the rate of uterine involution as defined in this study.

The objective of this study was to characterize the effect of dose and type of cloprostenol (CLO) on the luteolytic response of dairy cattle during the Ovsynch protocol under different oestrus cycle and physiological characteristics. Twelve non-lactating dairy cows and 111 lactating dairy cows were used in three experiments. In Experiment I, cows were synchronized so that they had only a 5.5- to 6-day-old corpus luteum (CL) at the time of the prostaglandin F_2α (PGF_2α) treatment of Ovsynch. In Experiment II, cows were synchronized so that they had at least a CL of approximately 14 days old at the time of PGF_2α treatment and an accessory CL if they had responded to the first GnRH of Ovsynch. Furthermore, in each experiment, cows received either a standard or a double dose of d-CLO as the luteolytic treatment. In Experiment III, lactating cows were blocked by parity and assigned to one of three luteolytic treatments during Ovsynch: 500 μg d,l-CLO, 150 or 300 μg of d-CLO. In Experiment I, the dose of d-CLO had an effect (p = 0.08) on the percentage of cows with full luteolysis, but not in Experiment II (p > 0.1). More cows in Experiment II had full luteolysis than did cows of Experiment I (87% vs 58%, respectively; p = 0.007). In Experiment III, 87.1%, 84.4% and 86.2% lactating dairy cows had full luteolysis and 37.8%, 36.8% and 36.1% of cows became pregnant after treatment with 500 μg d,l-CLO, 150 or 300 μg of d-CLO, respectively (p > 0.05).

In a conservation project, reproductive biotechnology was implemented for the recovery and conservation of an endangered bovine breed in Spain. The breed Murciana–Levantina, declared to be worthy of special protection status (http://www.fao.org/newsroom/en/news/2006/1000464/index.html), is of great interest because of its hardness, longevity, docility and disease resistance. This contribution describes the birth of the first calf of this breed obtained by reproductive biotechnology, using ultrasound-guided punction and aspiration of ovarian follicles, in vitro embryo production, vitrification of embryos by a cryotop device and, finally, the transfer of cryopreserved embryos to recipient heifers of a commercial dairy herd.

The aim of the present study was to examine the role of oxytocin (OT) in the progesterone (P4) and prostaglandins (PGs) pathway to induce oocyte meiotic resumption. Cumulus–oocyte complexes were co-cultured with follicular hemisections for 15 h to determine the effects of different doses of OT or atosiban (ATO; oxytocin receptor antagonist) on oocyte meiotic resumption. In another experiment, we examined the effect of the interaction between P4, OT and PGs on the regulatory cascade of the oocyte meiotic resumption. Oxytocin at 1 μm was effective in inducing meiotic resumption in oocytes co-cultured with follicular cells (84.0%), not differing from the positive control group (74.4%). Atosiban inhibited in a dose-dependent manner the positive effect of OT on the meiotic resumption (27.6% metaphase I with 10 μm of ATO, which did not differ from the 25.5% of the negative control group). Furthermore, a third experiment showed that P4 was able to induce oocyte meiotic resumption, which was inhibited by ATO. However, the OT positive effect was not blocked by mifepristone (P4 antagonist), but was inhibited by indomethacin (a non-selective PTGS2 inhibitor). Collectively, these data suggest a sequential role of P4, OT and PGs in the induction of oocyte meiotic resumption.

The regulation of granulosa cell proliferation is complex, and it is essential for normal follicular development in mammals. The aim of this study was to examine the expression of cyclins and their inhibitors in the granulosa cells of follicles at different developmental stages. Follicles were classified into three groups: oestrogen-inactive dominant follicles (EIDs), oestrogen-active dominant follicles (EADs) and pre-ovulatory follicles (POs). The expression of CCND2 (cyclin D2) mRNA was significantly higher in granulosa cells from EADs and POs than in those from EIDs. The expression of CCND3 (cyclin D3) mRNA was significantly higher in granulosa cells from EADs than in those from other follicles. CCND1 (cyclin D1), CCNE1 (cyclin E1) and CCNE2 (cyclin E2) mRNA expression did not differ among the different follicular stages. The expression of CDKN1A (p21^cip1) and CDKN1B (p27^kip1) mRNA was significantly higher in granulosa cells from EIDs and POs, respectively, than in those from other follicles. Expression of CDKN2D (p19^INK4d) mRNA did not differ among the different follicular stages. Taken together, our study suggested that cyclins and their inhibitors are associated with granulosa cell proliferation at specific follicular developmental stages.

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed.

Mycobacterium avium subspecies paratuberculosis (MAP), the causal agent of paratuberculosis, was detected by quantitative real-time IS900 PCR in the follicular fluid from the reproductive tracts of cows originating from one infected herd. As well as being detected in follicular fluid of cows shedding bacteria in their faeces, MAP was also detected in the follicular fluid of one apparently healthy, non-shedding individual cow. The finding of MAP in follicular fluid is unexpected and could contribute to the lower viability of embryos and resultant lower pregnancy rate. In addition to finding contaminated follicular fluid, vaginal and uterine flush fluids were determined to be positive for the presence of MAP in 75% and 56.3% of the time of the cattle currently shedding MAP in their faeces, respectively. The presence of MAP in different parts of the reproductive tract was seen in clinically as well as subclinically infected cows. These findings extend our currently scant and contradictory knowledge about the dissemination of MAP in the reproductive tract of female cattle.

Numerous experimental models in different species have been developed for the study of polycystic ovarian syndrome. In this study, we used a model of induction of polycystic ovaries (PO) in rats by exposure to constant light to study the distribution and variations of glycosylated residues present in the different ovarian structures. Seven biotinylated lectins were used (Con-A, WGA, DBA, SBA, PNA, RCA and UEA-I) on tissue sections, and detection was performed using the streptavidin/peroxidase method. In tissue sections was observed an increase in affinity for Con-A in the granulosa and theca interna of growing follicles and cysts in animals with PO in relation to the control group. Follicular cysts showed higher affinity for WGA and RCA-I which growing follicles in the same group, and there was a decrease in affinity for PNA in the cysts in relation to the growth of follicles in both groups. Atretic follicles in both groups showed greater labelling with lectins PNA, SBA and RCA-I in relation to healthy follicles. It could also be noted that the zona pellucida of cystic follicles lost the affinity for the lectin Con-A. There was no staining on follicles in any category with the lectins DBA and UEA-I, although it was staining in the corpus luteum (control group) and in the mesothelium and interstitial glands of both groups with DBA. These observations probably reflect changes in the glycosaminoglycans present in the different ovarian compartments or in the glycosylation of cellular components essential for proper follicular dynamics.

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8-cell, 16-cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16-cell and blastocyst stages, relative mRNA abundance of stress-related genes HSP 70.1 and HSP 70.2 and pro-apoptotic genes CASPASE-3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress-related gene HSF1. Expression level of anti-apoptotic genes BCL-XL and MCL-1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16-cell and blastocyst stages. Among the genes related to embryonic development, at 8–16-cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR-1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress-, apoptosis- and development-related genes.

The variation in the kidding size of Black Bengal and Sirohi breed of goats makes them an interesting genetic material to study the underlying genetic mechanism of prolificacy. Accordingly, we studied the comparative ovarian morphometry including disparity in numbers of antral follicles of different sizes between these two breeds. Further, we evaluated the differential expression of the important candidate genes (viz., BMP15, GDF9 and BMPR1B) known to influence the ovulation rates and the prolificacy. The ovaries of Black Bengal (n = 20) goat were lighter (p < 0.01) in weight and smaller (p < 0.01) in diameter than those of Sirohi (n = 19) goats but possessed more numbers (p < 0.05) of corpus luteum (CL), large and small antral follicles. Quantitative real-time PCR (RT-qPCR) analysis revealed differential expression of mRNAs encoding for the BMP15 and GDF9. Small antral follicles of Black Bengal goats expressed 2.78-fold more (p < 0.05) of BMP 15 than those of Sirohi goat. Expression of BMP15 (p < 0.01) and GDF9 (p < 0.05) mRNAs was more abundant in the small than the large antral follicles of Black Bengal goat. The more numbers of antral follicles per unit of ovarian mass and differential expression of BMP15 and GDF9 may serve as an important clue for higher prolificacy.

The study aimed to compare the acid–base balance and steroid concentrations between follicular fluids (FF) of pre-ovulatory follicles derived from a spontaneous oestrus (SO), synchronized or induced oestrus (IO) and follicular cysts (CYS) and between FF and blood in dairy cows. Forty-two dairy cows were included in this study. The animals were allocated to three groups: SO (n = 23); IO (n = 11) using GnRH at day 0 and day 9 and PGF₂α at day 7; and animals with CYS (n = 10). The follicular fluids (FF) were aspirated from the cyst/pre-ovulatory follicles (∅ ≥ 15 mm) after SO and after second GnRH dose in IO by transvaginal ultrasound-guided ovum pick-up technique. Blood samples (BL) were collected in heparinized vacutainer tubes. The oxygen tension (pO2) in FF of IO was higher (p < 0.05) than in SO and CYS groups. There were negative correlations (p < 0.001, r = −0.89) between FF and blood pO2. The carbon dioxide tension (pCO2) and lactate level in FF of CYS group were higher (p < 0.05) than in SO and IO groups. There were negative correlations (p < 0.01, r = −0.73) between blood and FF pO2. Oestradiol-17β concentration in pre-ovulatory follicles and plasma of the SO group was higher (p < 0.001) than in IO and CYS groups. Progesterone concentration in pre-ovulatory follicles and plasma of the SO and IO groups was lower (p < 0.01) than in CYS group. Plasma androstenedione concentration in SO and IO groups was higher (p < 0.05) than in CYS group. In conclusion, acid–base parameters, E2 and P4 levels in the follicular fluid of both IO and CYS groups were deviated greatly from the physiological level (disturbances of intrafollicular/intracystic environment), which may affect the quality of both the oocyte and the granulosa cells.

The aim of this study was to demonstrate that seroreactivity against Leptospira is significantly associated to the reproductive efficiency of recipient mares of an embryo transfer (ET) programme. A serosurvey was conducted from August 2007 to March 2009 in five herds from Rio de Janeiro, Brazil, with high rates of reproductive failure, as early embryonic death (>12%), abortion (>12%) and perinatal death. Detailed information about the losses was obtained from practitioner. A total of 338 recipient mares were tested by microscopic agglutination test, and 226 (66.9%) were seroreactive, mainly against serovars Bratislava and Copenhageni. Seroreactivity could be associated to reproductive failure (p < 0.001), and it was demonstrated that a seroreactive mare is 1.8 times more likely (relative risk – RR) to present reproductive failure than a seronegative one, particularly in relation to early embryonic death (p < 0.0001; RR 8.4) but also to abortions (p < 0.0001; RR 3.5), and to perinatal death (p < 0.05; RR 7.3). Therefore, seroreactivity to Leptospira is associated to reproductive failure in all phases of pregnancy in recipient mares, impairing equine ET programmes.

The aim of this study was to investigate the expression profile of candidate genes involved in competence during oocyte growth. The candidate genes (BMP15, OOSP1, H1FOO, H2A, H3A, H4, SLBP, DNMT1, DNMT3B, HAT1, HDAC2 and SUV39H1) were selected because of their possible involvement in determining oocyte developmental competence. Pre-antral and antral follicles were isolated from the ovaries of Zebu (Bos indicus) cows, measured and classified into the following categories according to their diameter: (i) oocytes from primordial follicles: diameter <20 μm, (ii) oocytes from primary follicles: 25–35 μm, (iii) oocytes from small secondary follicles: 40–60 μm, (iv) oocytes from large secondary follicles: 65–85 μm, (v) oocytes from small antral follicles: 100–120 μm, and (vi) oocytes from large antral follicles: >128 μm. Total RNA was extracted from four pools of 25 oocytes for each category of follicles, and the genes were quantified by qPCR. Target gene expression was normalized using the gene PPIA. The results suggest that stocks of the studied transcript genes accumulate before the final phase of folliculogenesis. The HDAC2 gene was the only gene in which a differential expression was observed at stage associated with competence acquisition.

Subinvolution of placental sites (SIPS) is the major cause of persistent sanguineous vaginal discharge after parturition in the bitch. Spontaneous remission is common but may take several months, and hence, medical therapy to end the discharge is often requested. In this retrospective study, we evaluated the effect of treatment for SIPS with low oral doses of a progestagen. Nine bitches with SIPS, but otherwise clinically healthy, were found in the computer database of the Department of Clinical Sciences of Companion Animals. Seven of these bitches were treated with low oral doses of a progestagen (megestrol acetate, 0.1 mg/kg body weight (bw) once daily for the 1st week, then 0.05 mg/kg bw once daily for the 2nd week). The other two bitches were untreated. Treatment results were evaluated by a telephone questionnaire. Progestagen treatment was successful in all of the treated dogs; sanguineous vaginal discharge stopped within the treatment period. One of the two untreated dogs remained symptomatic until the next oestrus, approximately 120 days after parturition, and the other remained symptomatic until 6 weeks before the start of the next pro-oestrus, 270 days after parturition. No side effects of the progestagen treatment were observed. Subsequent gestations, parturitions and puerperal periods of 5 mated bitches were uneventful. One bitch did not become pregnant after mating. In conclusion, the results of this study indicate that oral administration of low doses of progestagen for 2 weeks is effective in stopping persistent sanguineous vaginal discharge in bitches with SIPS, with neither side effects nor reduced subsequent fertility.

Priapism, a persistent long-lasting involuntary erection of the penis, is uncommon in dogs. In this report, the case of a 13-year-old male Pointer, referred to our services due to persistent exposition of the penis, is described. This condition was consecutive to an intermittent priapism situation lasting for several days, which has been initially attributed to the inflammation and haematoma associated with a perianal bite. The owners became unable to retract the penis into the prepuce. At presentation, the dog was anorectic for 48 h, intolerant to manipulation, and showed poor body condition and unsteady locomotion. During physical evaluation, a marked engorgement of the local vessels in the prepuce and penis was found. An abdominal X-ray was asked under the suspicion of a neurogenic origin for the clinical situation, which showed evidences of spondylosis. After discussion of the clinical condition, the owners asked for euthanasia. The necropsy confirmed the engorgement of the regional vessels deriving from the pudendal arteries and blood accumulation within all the cavernous spaces, accompanied by congestion and thrombosis within the erectile structures of the penis. No significant changes were observed in the pelvic organs that could be at the origin of priapism. The lumbar-sacral spinal regions were carefully inspected and evidenced signs of L7-S1 stenosis due to spondylosis. The case presented herein is a rare situation of priapism of neurogenic origin in a dog. Necropsy findings suggest that it was consecutive to cauda equina compression due to lumbar spinal stenosis.

Interferon tau (IFNT), a type I IFN produced by the conceptus trophectoderm, is the signal for maternal pregnancy recognition in ruminants. The purpose of this study was to investigate whether IFNT effected on the proliferation of ovine trophectoderm cells in an autocrine manner. Elongated ovine conceptuses (Days 15, Day 0 = day of mating) were collected for isolation of mononuclear ovine trophectoderm (oTr-1) cells, and conceptuses (Days 15 and 20, n = 4 and 3, respectively) were collected for RNA extraction. We demonstrated that the IFNT receptor, IFNAR1, was expressed in trophectoderm of day 15 and 20 conceptuses. Interestingly, the ovine trophectoderm cell line oTr-1 cultured in the presence of recombinant bovine IFNT (rbIFNT) displayed increased expressions of IFN-stimulated genes (ISGs), such as IFN-stimulated gene 15 (ISG15), 2-5-oligoadenylate synthetase 1 (OAS1) and bone marrow stromal cell antigen 2 (BST2). Meanwhile, the presence of rbIFNT in the culture media could promote the cell proliferation in a dose-dependent manner. Furthermore, the connective tissue growth factor, which has diverse functions in cell proliferation and is involved in conceptus elongation, was upregulated in oTr-1 cell by rbIFNT treatment in vitro. These data indicated that IFNT could act as an autocrine factor to regulate trophectoderm cell proliferation.

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid-luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non-angiogenic function.

The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. The results of basic tests did not demonstrate significant differences amongst stallions, except for progressive sperm motility, which was significantly higher (p < 0.05) in the semen of Arabian stallions. In the semen of the same stallions, a significant decrease in the percentage of alive spermatozoa was observed at 72, 96 and 120 h of storage, whereas a significant increase in the number of spermatozoa with DNA defragmentation was found after 24 h storage. In the semen of coldblood stallions, significantly reduced live spermatozoa percentage was observed at 96 and 120 h, while increased DNA defragmentation was observed at 48 h. These findings demonstrated that the semen of Arabian stallions chilled to +4°C retained original characteristics until 24 h of storage, whereas in coldbloods, these were preserved up to 48 h of storage.

This study was performed to evaluate the structural preservation of antral follicles after bovine ovarian tissue vitrification using histological analysis. Ovaries (n = 30) of slaughtered cows were cut into small fragments using a scalpel blade, and the ovarian tissues were randomly assigned to vitrification using 15% dimethyl sulphoxide (DMSO) and 15% ethylene glycol (EG) and fresh tissues (control) groups. For histological evaluations, fresh and post-thawing ovarian tissues were immediately fixed, serially sectioned into 5-μm sections and stained with haematoxylin and eosin (H&E). Nine serial sections per fragment were subjected for morphological assessment. The diameter of the antral follicles was determined and classified into four groups: 1 (≤1 mm), 2 (>1–2 mm), 3 (>2–3 mm) and 4 (>3–4 mm). Then, follicular morphology was evaluated in relation to atresia and categorized into seven grades: Grade A (healthy follicle); Grades B, C and D (early atresia); Grades E and F (moderate atresia); and Grade G (advanced atresia). The results revealed that small diameters of antral follicles (1 and 2 mm) were more susceptible for cryoinjury. The normal follicular morphology (Grade A) was not affected by vitrification throughout follicle diameters. Nevertheless, some damage features were monitored after vitrification. In conclusion, the morphological structure of bovine antral follicles could be successfully preserved by ovarian tissue vitrification.

The corpus luteum (CL) of the pig lacks luteolytic sensitivity (LS) to prostaglandin (PG) F-2α until after day 12 of the oestrous cycle, but the mechanisms underlying this phenomenon are poorly understood. As luteolysis involves apoptosis, we hypothesized that critical apoptotic proteins may be deficient in CLs that lack LS. The specific aim of these studies was to examine mRNA expression and protein levels of apoptosis genes/proteins (BAX/Bax, BCLX/Bcl-x, CASP3/Caspase-3, CASP8/Caspase-8, NFΚB1/NFκB, TP53/p53) in porcine CLs collected at different stages of the oestrous cycle. CLs were collected surgically, mRNA and protein extracted, and expression/levels analyzed by semi-quantitative (SQ) PCR and Western blots, respectively. At the mRNA expression level, only BAX (maximal on day 4) and TP53 (maximal on day 7) showed significant variations during the oestrous cycle. At the protein level, only Bcl-x and Caspase-3 showed significant changes during the cycle; Bcl-x decreased on day 13 and Caspase-3 increased on day 13. It is concluded that apoptosis-associated proteins (i.e. Bcl-x and Caspase 3) may play a critical role in luteolytic sensitivity in the pig.

This study was carried out with the objectives to test the differences in the haemodynamic characteristics of the prostatic artery in normal and benign prostatic hyperplasia (BPH)–affected dogs using Doppler ultrasonography. In sixteen male German shepherd dogs, prostatic volume was detected and prostatic biopsy was performed. The prostatic artery blood flow parameters determined were as follows: peak systolic velocity (PSV), end diastolic velocity (EDV), Resistive Index (RI) and Pulsatility Index. The power Doppler mode was used for colour flow mapping of the prostatic artery. In PW mode at marginal locations, the waveforms recorded showed a continuous pattern typical of the high-resistance vessels, while in subcapsular locations, the waveforms recorded were continuous characteristic of the low-resistance vessel. Peak systolic velocity and EDV in both locations were significantly higher in BPH group (p < 0.001) than normal group (p < 0.05). Pixel number in BPH group in both locations was significantly higher (p < 0.05) compared to the normal group. This study shows that Doppler ultrasonography represents a valid and non-invasive method for the characterization of the blood flow in the prostatic artery in dogs affected with BPH. Moreover, statistically significant differences of blood flow velocities in prostatic artery in normal and BPH-affected dogs were detected. The RI was not able to differentiate normal dogs from dogs affected by BPH and therefore is not a parameter usable for diagnostic purposes, while Power Doppler could represent an additional diagnostic tool.

The development of antral ovarian follicles entails fluid accumulation, but the mechanisms regulating water flux are unknown. Aquaporins are small, integral membrane proteins facilitating passive movement of water, some of which are known to be regulated by steroid hormones. The aim of this study was to determine whether testosterone (T) influences water transport in porcine granulosa cells. To assess water movement, the swelling of granulosa cells when moved from isotonic (319 mOsm) to hypotonic (95 mOsm) medium was measured after 12-hour pre-incubation in the presence of either testosterone (T), the antiandrogen 2-hydroxyflutamide (HF) or HF and T together. Pre-incubation with T increased the swelling of granulosa cells (p < 0.01) and this was abolished by HF (p < 0.001). Neither T nor HF affected cells in isotonic medium (319 mOsm). The results indicate that T acting via intracellular androgen receptors increases water permeability of porcine granulosa cells, probably through the regulation of aquaporin activity.

This study investigates the association of semen traits with boar fertility. The fertility outcome (farrowing rate – FR and total piglets born – TB) of 14 boars was obtained from a field trial conducted during 10 week of breeding period on a commercial farm using multiparous sows (n = 948) through single-sire mating with 2 × 10⁹ motile sperm cells per artificial insemination (AI) dose. Sperm motion parameters, evaluated with computer-assisted semen analysis system in raw and stored semen at 17°C for 240 h, in addition to morphological sperm defects, measured on the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate cluster, canonical and discriminant analyses. Four clusters of boars were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The other boars were separated in two distinct clusters (four and seven boars), including boars with intermediate TB (12.64 and 13.22) but divergent values for FR (95.9% vs 91.8%). Semen traits with higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement and cytoplasmatic droplets. Through multivariate discriminant analysis, more than 80% of the 140 ejaculates were correctly classified into their own group, showing that this analysis may be an efficient statistical tool to improve the discrimination of potential fertility of boars. Nevertheless, the validation of the relationship between fertility and semen traits using this statistical approach needs to be performed on a larger number of farms and with a greater number of boars.

The pig exhibits a non-invasive, epitheliochorial placentation. Adhesion molecules are indispensable for successful implantation and establishment of placentation. CD34 is an adhesion molecule belonging to the immunoglobulin superfamily (IgSF). To take the first step to investigate the role of CD34 in placentation, we examined the expression pattern of CD34 at the maternal–foetal interface in Yorkshire gilts on days 15, 26, 50 or 95 and in Meishan gilts on days 26, 50 or 95 of pregnancy (n = 3 gilts/breed/day of pregnancy) by immunohistochemical technique. The CD34-positive signals were detected in uterine luminal epithelium and trophectoderm in Yorkshire pigs; the staining for CD34 was located in trophectoderm but barely detectable at the uterine luminal epithelium on day 15 of pregnancy. Then, the expression of CD34 increased dramatically in both the uterine luminal epithelium and trophectoderm by day 26, and weak staining intensity was observed at the maternal–foetal interface on days 50 and 95 of pregnancy. The expression pattern of CD34 in Meishan pigs is similar to that in Yorkshire pigs except that only a few positive signals were observed at the luminal epithelium on day 26 of pregnancy. These results suggest that CD34 may be involved in mediating the cell-to-cell adhesion between trophectoderm and the luminal epithelial cells during early pregnancy in pigs.

Pre-natal glucocorticoids are used in women at risk of preterm delivery to induce foetal lung maturation. However, glucocorticoids can produce negative outcomes for other tissues such as the reproductive system. We therefore tested the effects of pre-natal betamethasone on testicular morphology and apoptotic protein immune expression during pre- and post-natal development. Pregnant ewes (n = 42) bearing singleton male foetuses were randomly allocated to receive intramuscular injections of saline or betamethasone (0. 5 mg/kg) at 104, 111 and 118 days of gestation (DG). Testes were collected at 121 and 132 DG, and at 45 and 90 post-natal days (PD) and subjected to morphometric analysis (volume densities of sex cords and interstitial tissues; sex cord diameter). Immunohistochemistry (% stained area) was used to assess active caspase-3, Bax, Bcl-2 and cell-cycle proteins (PCNA). Compared with control values, betamethasone treatment decreased sex cord diameter at 121 DG, 45 and 90 PD, and sex cord volume at 90 PD. Active caspase-3 was decreased by betamethasone at 121 DG and 90 PD, but Bax was increased in all betamethasone groups. Bcl-2 and PCNA decreased in the betamethasone groups at 121 DG and 45 PD, but increased at 132 DG and 90 PD. We conclude that high levels of pre-natally administered glucocorticoid reduce foetal testicular development, perhaps via changes in the balance between pro- and anti-apoptotic proteins and cell-cycle proteins. These outcomes could compromise the future spermatogenic potential of male offspring.

Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10⁻¹², 10⁻⁹, 10⁻⁴ m; Experiment 4: 0, 10⁻³ m), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat-stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10⁻⁴ m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non-HSO without melatonin. Melatonin did not have any effect in embryos from non-HSO groups compared with the control. In Experiment 4, 10⁻³ m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non-HSO when compared to melatonin-untreated oocytes/embryos. In conclusion, 10⁻⁴ m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase-2 (COX-2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post-GnRH. Blood samples were collected daily for progesterone and estradiol-17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX-2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol-17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down-regulated until its lowest expression was reached on day 10 post-GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX-2 was highest from day 8 to 12 post-GnRH in the luminal epithelium, in relation to the time of maximal PGF_2α release. Both steroid receptors populations and COX-2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8–10 post-induction of ovulation and the concomitant increase of COX-2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.

PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p < 0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures.

Ewes heterozygous for the FecX^R allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecX^R allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild-type (++) ewes were subjected to 2 laparoscopic ovum pick-up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH-treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (−0.2 mm in untreated and −0.8 mm in FSH-treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus–oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture.

Melatonin is thought to be the main molecule that transmits the signal of seasonal change to the neuroendocrine system in seasonal breeding species. Melatonin exerts its effects through specific melatonin receptors, MTNR1A and MTNR1B. In the present study, six native goat breeds in China and one introduced goat breed were analysed to investigate the relationship between the genetic polymorphism of receptor genes and seasonal reproduction. Sequencing results showed that there were five polymorphic mutations in the MTNR1A gene and two in the MTNR1B gene. In the MTNR1A gene, genotypes AA, AB and BB for 424C>T and genotypes CC, CD and DD for 589C>A were observed in these goat breeds. In all six native goat breeds, only genotype AA was detected. In the MTNR1B gene, genotypes EE, EF and FF for 1179G>A and genotypes GG, GH and HH for 1529A>G were detected. However, in Gulin Ma goats, the genotypes EE and HH were not found. Moreover, the base of G at position 1179 and A at position 1529 were linked (By Arlequin ver 3.1, Zoological Institute, Berne, Switzerland, http://cmpg.unibe.ch/software/arlequin3,D′ = 0.7496, r² = 0.4421, χ² = 489.8679, p = 0.000). Among these mutations, no amino acid change was found in MTNR1A, while both of the mutations in MTNR1B gene caused amino acid changes of R222H and S339G, respectively. The structural analysis showed that the R222H mutation occurred in the first amino acid residue of the third cytoplasmic loop, and the S339G mutation was located in the carboxyl terminus of the protein. In terms of seasonal breeding, all the genotypes we detected showed a similar kidding frequency distribution trend with a higher frequency in May–August than in January–April and in September–December. This suggests that the relationship between the polymorphisms in the MTNR1A and MTNR1B genes and seasonal breeding could not be established.

The maintenance of antioxidative/oxidative balance is crucial for cellular and extracellular environment. That is why antioxidative enzymes express their activity in different isoforms in different cell compartments and extracellular space. The aim of study was to verify the results of previous experiment on activities of antioxidative enzymes by the determination of their enzymatic proteins in bovine placental tissues by Western blotting technique. Moreover, the presence of particular isoenzymes was detected and differentiated. Homogenates of maternal and foetal part of both properly released and retained bovine placenta were subjected to PAGE electrophoresis in non-reducing and reducing conditions and Western blotting with appropriate antibodies against superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Electrophoresis allowed for the detection of protein bands of molecular weight related to CuZn-SOD as well as cGSH-Px isoenzymes. The reaction with appropriate antibodies confirmed this. Densitometric analysis, although semi-quantitative, allowed for the observation of trends in differences in antioxidative enzyme proteins, which may partly confirm previously described results in cases of retained and released placenta. Local antioxidative enzymatic mechanisms in bovine placental tissues are represented by CuZn-SOD and cGSH-Px, which show the changes in their expression during improper placental release.

The mechanisms that regulate the apoptosis are essential to the normal development and maintenance of homoeostasis and play an important role in placental development in mammals. During porcine pregnancy, there must be a proper cellular remodelling to achieve a normal gestational development. Knowledge of pig physiology during pregnancy will explore options to increase the productivity of this species of high economical value. The purpose of this work was to study the cell morphology and apoptosis of porcine placentas from early, mid and late pregnancy. For that purpose, high-resolution light microscopy and transmission electron microscopy were performed to the study of cell morphology. TUNEL, the apoptosis index (IAp) and the expression of c-FLIP through immunohistochemistry technique were used to the study of apoptosis. High-resolution light microscopy and transmission electron microscopy confirmed the presence of placental cells with ultrastructural apoptotic features. Apoptotic nuclei were detected by TUNEL in different placental structures and phagocytes containing apoptotic bodies. The IAp in villi was 9.34% at early, 0.82% at mid and 23.85% at late pregnancy. Statistically significant differences were found between periods (p < 0.05). In previous studies, we determined a differential induction of the apoptotic routes in the placental villi in agreement with the gestational period. A co-expression of receptors and mitochondrial proteins in placental connective tissue was detected, but the immunolocalization of c-FLIP would indicate an endogenous inhibition of the extrinsic pathway. In conclusion, in swine there exists differential activation of inducing apoptotic pathways in different placental structures according to the gestational period.

Although thyroid dysfunction occurs frequently in humans and some animal species, the mechanisms by which hypo- and hyperthyroidism affect the corpus luteum have not been thoroughly elucidated. This study evaluated the levels of proliferative activity, angiogenesis, apoptosis and expression of cyclooxygenase-2 in the corpus luteum of female rats with thyroid dysfunction. These processes may be important in understanding the reproductive changes caused by thyroid dysfunction. A total of 18 adult female rats were divided into three groups (control, hypothyroid and hyperthyroid) with six animals per group. Three months after treatment to induce thyroid dysfunction, the rats were euthanized in the dioestrus phase. The ovaries were collected and immunohistochemically analysed for expression of the cell proliferation marker CDC-47, vascular endothelial growth factor (VEGF), VEGF receptor Flk-1 and cyclooxygenase-2 (COX-2). Apoptosis was evaluated using the TUNEL assay. Hypothyroidism reduced the intensity and area of COX-2 expression in the corpus luteum (p < 0.05), while hyperthyroidism did not alter COX-2 expression in the dioestrus phase. Hypothyroidism significantly reduced the expression of CDC-47 in endothelial cells and pericytes in the corpus luteum, whereas hyperthyroidism did not induce a detectable change in CDC-47 expression (p > 0.05). Hypothyroidism reduced the level of apoptosis in luteal cells (p < 0.05) and increased VEGF expression in the corpus luteum. In contrast, hyperthyroidism increased the level of apoptosis in the corpus luteum (p < 0.05). In conclusion, thyroid dysfunction differentially affects the levels of proliferative activity, angiogenesis and apoptosis and COX-2 expression in the corpus luteum of female rats.

The aryl hydrocarbon receptor (AHR) is an intracellular transcription factor best known for mediating the toxicity of dioxins. The AHR is phylogenetically highly conserved among invertebrates and vertebrates and may play a significant role in the regulation of physiological processes including female reproduction. This study was performed to determine the partial nucleotide sequence of porcine AHR and to evaluate the AHR mRNA (real-time PCR) and AHR protein (Western blot) expression in granulosa and theca interna cells harvested from medium (3–6 mm) and pre-ovulatory (≥8 mm) follicles as well as in luteal cells obtained from corpora lutea collected during the mid-luteal phase (days: 8–10) of the porcine oestrous cycle. In the study, the partial nucleotide sequence of porcine AHR containing 1021 bp (GenBank accession no: HM488957.1) was determined. The AHR transcript and protein were found in all ovarian cells obtained during both phases of the porcine oestrous cycle. The highest AHR transcript level was detected in theca interna cells isolated from pre-ovulatory follicles as well as in luteal cells. Higher AHR protein expression was found in granulosa cells isolated from pre-ovulatory follicles in comparison with all remaining cell types. The presence of AHR in the examined ovarian cells may account for their sensitivity to some environmental pollutants. Moreover, the differences found in AHR mRNA expression between granulosa and theca cells as well as between cells originating from follicles of different size suggest the involvement of AHR in the modulation of reproductive processes in the porcine ovary.

The osteopontin gene may influence the fertility of water buffaloes because it is a protein present in sperm. The aim of this work was to identify polymorphisms in this gene and associate them with fertility parameters of animals kept under extensive grazing. A total of 306 male buffaloes older than 18 months, from two farms, one in the state of Amapá and the other in the state of Pará, Brazil were used in the study. Seven SNPs were identified in the regions studied. The polymorphisms were in gene positions 1478, 1513 and 1611 in the region 5′upstrem and positions 6690, 6737, 6925 and 6952 in the region amplified in intron 5. The SNPs were associated with the traits, namely scrotal circumference, scrotal volume, sperm motility, sperm concentration and sperm pathology. There were significant SNPs (p < 0.05) for all the traits. The SNP 6690 was significant for scrotal circumference, sperm concentration, sperm motility and sperm pathology and the SNP 6737 for scrotal volume. The genotype AA of SNP 6690 presented the highest averages for scrotal circumference, sperm concentration and motility and the lowest total number of sperm pathologies. For the scrotal volume trait, the animals with the largest volume were correlated with the presence of the genotype GG of SNP 6737. These results indicate a significance of the osteopontin gene as it seems to exert a substantial influence on the semen production traits of male buffaloes.

In six German Shepherds dogs, GnRH agonist implants (Deslorelin) were inserted subcutaneously one month after histological confirmation of benign prostatic hyperplasia (BPH). Prostatic volume (PV), characteristics of ejaculate, serum testosterone concentrations and Doppler parameters of prostatic and subcapsular arteries were detected at different time intervals, for 6 month. The prostatic volume showed a significantly reduction starting at day 37. The decrease in sperm concentration, motility and increase in morphological abnormal sperm were observed from day 22 to day 37, when it was no longer possible to obtain the ejaculate. The values of peak systolic velocity and end-diastolic velocity in prostatic and subcapsular arteries showed from day 11 a gradual decrease, significant at day 22 until day 37 and reaching the lowest values at day 52 until the end of observation. The power Doppler pixel intensity of both arteries showed a gradual decrease from day 5 until day 52. In particular, a significant decrease was observed for both arteries from day 11. Testosterone serum concentration decreased to undetectable levels by day 11 until the end of the observations. All these Doppler parameters and testosterone values were positively correlated with the prostatic volume. Furthermore, testosterone values were positively correlated with peak systolic velocity, end diastolic velocity and pixel numbers. The use of implants containing GnRH analogues, even in asymptomatic subjects, is effective for the control of BPH and the application of Doppler exam of prostatic blood flow represent an non-invasive tool for monitoring the response of medical treatment.

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS–egg yolk–based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25-ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0- and 6-h post-thaw. Experiment 1 compared the effect of three non-feline commercial extenders – based on TRIS–egg yolk (Triladyl), egg-yolk-free medium (AndroMed) and skimmed milk-egg yolk (Gent) – on the quality of frozen-thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen-thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS–egg yolk–based extenders, one non-feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing-thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing-thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS–egg yolk–based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.

Using a novel in vivo model considering a low developmental competence embryo (demi-embryo) and a subnormal fertility recipient (lactating high-yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy-specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi-embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG-treated cows with secondary CL than in hCG-treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG-treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non-pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi-embryos and the pregnancy rate of high-yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.

This study examined the effects of supplementation of ES-like cell culture medium with bone morphogenetic protein (BMP)-4 (0, 10, 20 or 100 ng/ml) or Noggin (250, 500 or 750 ng/ml) or TGF-β1 (0, 0.1, 1 or 10 ng/ml) or SB431542 (0, 10, 25 or 50 μm), an inhibitor of TGF-β1 signalling, on survival, colony area and expression level of pluripotency genes in buffalo ES-like cells at passage 40–80, under different culture conditions. BMP-4 supplementation significantly reduced (p < 0.05) colony survival rate, percentage increase in colony area and relative mRNA abundance of OCT4, whereas that of NANOG and SOX-2 was increased significantly (p < 0.05). Noggin supplementation did not affect the colony survival rate and percentage increase in colony area in the presence of FGF-2 and LIF. In the presence of FGF-2 alone, it significantly reduced (p < 0.05) the relative mRNA abundance of OCT4 and SOX-2 and increased (p < 0.05) that of NANOG. Supplementation with TGF-β1 at 1.0 ng/ml but not at other concentrations increased colony survival rate but had no effect on percentage increase in colony area at any concentration. Supplementation with SB-431542 decreased (p < 0.05) colony survival rate at 50 μm but not at other concentrations. The percentage increase in colony area was lower (p < 0.05) with 10 μm SB-431542 than that in the controls, whereas at higher concentrations of 25 or 50 μm, SB-431542 decreased (p < 0.05) the colony size instead of increasing it. In conclusion, these results suggest that BMP-4 induces differentiation in buffalo ES-like cells, whereas TGF-β/activin/nodal pathway may not be playing a crucial role in maintaining pluripotency in these cells.

Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p < 0.05). The pregnancy rates at 90 and 240 day were significantly lower in groups F2 (4.9% and 3.3%) and F3 (5.4% and 5.4%) compared to groups F1 (23.3% and 16.3%), F4 (25.7% and 18.6%) and F5 (25.9% and 19.8%) (p < 0.05). The cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p < 0.05). Moreover, large offspring syndrome (LOS) incidence in group F5 was significantly lower than those in other groups (p < 0.05). All cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves.

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR-14; Hoechst-33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR-14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane-affected sperm (semen treated with three cycles of freezing to −20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma-intact sperm determined by acridine orange and SYBR-14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.

The objective of the present study was to elicit opinion from two groups of veterinarians [subject matter experts and non-subject matter experts] about the causes of bovine perinatal mortality and the criteria used to assign such causes. The subject matter experts were selected on the basis of their scientific publications or experience of working in a veterinary diagnostic or research laboratory in the area of bovine perinatal mortality. The non-subject matter experts were self-selected as cattle veterinarians without particular expertise in bovine perinatology. A total of 74 veterinarians (46 subject matter experts and 28 non-subject matter experts) from 23 countries responded. The study was conducted using Delphi methodology over seven rounds. Respondents were asked to agree the causes of bovine perinatal mortality and for each cause to agree the supporting diagnostic criteria. There was a close agreement between groups on 16 causes of death apart from intra-uterine growth retardation (IUGR) and micronutrient imbalances which were accepted by fewer subject matter experts. There was inter-group consensus on the criteria to diagnose accidents, congenital defects, dystocia, hyperthermia, infections, premature placental separation, prematurity and prolonged calving. There was inter-group consensus on the criteria to diagnose anoxia, apart from gingival cyanosis; on haemorrhage, apart from haemorrhagic anaemia; on IUGR, apart from organ weights; and on iodine imbalance, apart from goitre and thyroid iodine content. The results from this study highlighted the current lack of standardization of the criteria used to define the cause of death for bovine perinatal mortality and the need for such standardization.

The liver is an important organ that contributes to milk production in dairy cows. The aim of this study was to examine whether liver conditions affect the characteristics of blood plasma and follicular fluid (FF) and whether supplementing in vitro maturation medium with FF from either cows with damaged livers (DL) or those with healthy livers (HL) affects oocyte developmental competence. Biochemical characteristics of FF were significantly correlated with those in plasma. As such, the characteristics of both plasma and FF were similarly affected by liver conditions in that the concentrations of total protein and inorganic phosphorus were higher for the DL cow group than for the HL cow group, whereas the concentrations of albumin, lactate dehydrogenase and calcium were lower for DL cows than for HL cows. In addition, supplementing the medium with DL-FF retarded the progression of the nuclear maturation of oocytes collected from the HL cows. On culturing oocytes in maturation medium containing HL-FF, DL-FF or foetal calf serum, the highest developmental rate to the blastocyst stage was observed in the HL-FF group, while the lowest developmental ratio was observed in the DL-FF group. The growth factor array of the FFs revealed that 10 growth factors were significantly downregulated in the DL-FF compared with those in HL-FF. In conclusion, the characteristics of plasma and FF are affected by liver conditions in a similar way. Concentrations of several growth factors were low in DL-FF, as was the ability of DL-FF to support oocyte maturation compared with that of HL-FF.

In the present study, we analysed the effect of fixative, breed, luteal stage and location on the nuclear density, volume density of connective tissue and vascular tissue/lumina within a bovine luteal gland in view of the development of an in vivo sampling technique to longitudinally monitor luteal histophysiology. The inner zone defined as the zone geometrically closest to the centre of the gland shows a significantly lower nuclear density (for all cell types) and a higher volume density of collagen fibres and vessels when compared with the outer zone (p < 0.001). The nuclear density in luteal glands from Holstein-Friesian cows is not significantly different from that in Belgian Blue cows, nor is it in stage II vs stage III glands. The collagen fibre content was significantly lower in glands of Belgian Blue cows (p = 0.01) and in younger glands (p = 0.003). Hence, it seems that the lower nuclear density in the inner zone was compensated by a higher amount of collagen fibres. As the type of fixative applied has a significant effect on the nuclear density of the different cell types, the present study warrants future research to further optimize the fixation protocol. As a conclusion, we can state that the topographic difference in nuclear distribution for the different cell types in a bovine luteal gland is only significant when comparing the inner vs the outer zone. This implies that if a sample representative for the whole gland has to be taken, for example, when taking an in vivo sample, it is necessary that the biopsy goes through the inner zone and contains the total diameter of the gland.

This study was carried out to compare the post-thaw cryosurvival rate and the level of apoptosis in vitro produced zona-free cloned buffalo blastocysts subjected to slow freezing or vitrification in open-pulled straws (OPS). Zona-free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re-expansion rate following post-thaw culture for 22–24 h. The post-thaw re-expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen-thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non-cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non-cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona-free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.

The objective of this study was to determine the duration for which sperm from the North American bison (Bison bison) could be chilled prior to being cryopreserved, without compromising post- thaw sperm quality. This would permit transport of samples collected remotely, to the laboratory (at 4°C) for cryopreservation. Epididymal sperm from plains bison (n = 11) and ejaculated sperm from wood bison (n = 3) were collected, extended and held at 4°C for extended periods of time. At intervals, an aliquot was cryopreserved. Post-thaw sperm motion characteristics were evaluated by computer assisted sperm analysis. Representative plains bison sperm samples (n = 3) were evaluated for their in vitro fertilizing ability in a heterologous system using bovine oocytes. There was no statistical difference in total and progressive motility of plains bison epididymal sperm when cryopreserved after chilling for 24, 48 or 72 h. For wood bison ejaculated sperm, there was no difference in total and progressive motility for sperm cryopreserved following 24 or 48 h of chilling. However, one of the three bulls showed significantly poorer fertilization (based on cleavage rate) with sperm chilled for 72 compared to 24 and 48 h prior to freezing. In conclusion, plains bison epididymal sperm can be chilled for 72 h and wood bison ejaculated sperm can be chilled for at least 48 h prior to cryopreservation without compromising post-thaw sperm motility, while heterologous in vitro fertilization (IVF) assay indicated a between-bull variation in the in vitro fertilizing ability of sperm chilled for an extended duration before cryopreservation.

The present study sought to determine: (i) the effects of Neospora caninum infection and twin pregnancy on plasma pregnancy-associated glycoprotein-2 (PAG-2) concentrations throughout pregnancy and (ii) whether plasma PAG-2 concentrations could predict abortion in N. caninum-infected cows. The study was performed on a commercial Holstein-Friesian dairy herd in northeastern Spain and the final data included those recorded in 53 non-aborting and 19 aborting animals. Blood samples were collected immediately before pregnancy diagnosis (on Days 40, 90, 120, 150, 180 and 210 post-insemination) in non-aborting cows or until the time of abortion detection in aborting cows. General lineal models (GLM) repeated measures anova revealed the different behaviour of PAG-1 and PAG-2, and significant effects of Neospora seropositivity, cool season and twin pregnancy on plasma PAG-2 concentrations throughout gestation (between-subject effects). In addition, based on the odds ratios, the likelihood of abortion increased in Neospora-seropositive cows (by a factor of 7.0) compared to seronegative animals and decreased in cows with a high plasma PAG-2 concentration (>4.5 ng/ml) on Day 120 of pregnancy (by a factor of 0.24), compared to the remaining cows. In conclusion, there is a relationship between plasma PAG-2 concentrations and the risk of abortion in Neospora-infected dairy cows. Thus, plasma PAG concentrations measured using anti-boPAG-2 antiserum on Day 120 of gestation could serve as an indicator of the abortion risk in N. caninum infected animals; values <4.5 ng/ml indicating a high risk of abortion in chronically infected animals.

The first objective of this study was to evaluate intrauterine nitric oxide (NO) and endometrial inducible NO synthase (iNOS) in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) within 24 h after breeding. Mares susceptible (n = 6) or resistant (n = 6) to PBIE were inseminated over five cycles, and uterine secretions and endometrial biopsies were collected before and 2, 6, 12 and 24 h after insemination. Uterine secretions were analysed for NO and biopsies were analyzed for iNOS expression. A second experiment evaluated the effect of treatment with dexamethasone or mycobacterial cell wall extract (MCWE) on uterine NO production and endometrial iNOS mRNA expression. Six susceptible mares were inseminated over three cycles with (i) killed spermatozoa without treatment (control), (ii) killed spermatozoa with 50 mg of dexamethasone IV or (iii) MCWE IV 24 h prior to insemination with killed spermatozoa. Six resistant mares were inseminated with killed spermatozoa as a control. Six hours after breeding, uterine biopsies and secretions were collected and evaluated for NO and iNOS mRNA. In Experiment 1, resistant mares had an increase in iNOS mRNA expression 2 h post-breeding compared to baseline (p = 0.045), 12 h (p = 0.014) and 24 h (p = 0.001). Susceptible mares had higher expression 2 h compared to 6 h (p = 0.046). No differences were observed in mRNA or protein expression of iNOS between resistant and susceptible mares. Resistant mares had a relatively steady amount of total intrauterine NO over 24 h, while susceptible mares had an increase over time, with a significantly higher increase in total NO than resistant mares at 6 (p = 0.04) and 12 h (p = 0.032). In Experiment 2, no differences were observed for iNOS mRNA expression. Susceptible mares had increased NO when compared to resistant mares (p = 0.008) and MCWE decreased NO (p = 0.047).

In recent years, intensive attention has been put on improving reproductive performance of pigs. Several experiments aimed to identify markers associated with prolificacy, but this issue still remains open. In our study, we investigated associations between polymorphisms in IGF2, GNAS and MC4R genes with reproductive traits of Polish Landrace and Large White pigs. We did not find any significant associations for g. GNAS314T > 324C, IGF2 intron3-g.3072G > A or g. MC4R 1426G > A in Polish Landrace and Large White pigs. In the case of IGF2 intron3-g.3072G > A, this information is of great importance, because this marker is widely implemented in pigs breeding and previous experiments suggested its role in prolificacy of pigs. We also investigated expression of IGF2 gene and showed that this gene is monoallelically expressed in reproductive organs (ovary and cornus uteri).

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, a species-specific colloid were evaluated for their ability to separate spermatozoa from virus in ejaculates from carrier stallions. The three types of SLC significantly reduced the virus titre in fresh semen at 0 h and in stored semen at 24 h (p < 0.001) but did not completely eliminate the virus. Sperm motility parameters such as total motility and progressive motility were significantly increased after colloid centrifugation, whereas curvilinear velocity and amplitude of lateral head deviation were decreased, and the remainder (straight line velocity, average path velocity, straightness, linearity, wobble and beat cross-frequency) were not significantly affected by the processing. Although virus titres were reduced in the SLC samples, significant levels of infectivity still remained, especially in stallions shedding large amounts of virus. It remains to be determined whether SLC-processed sperm samples from stallions shedding low virus titres retain sufficient equine arteritis virus to cause infection in mares through artificial insemination.

In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC-treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.

The aim of this study was to determine the effect of melatonin treatment during the early dry-off period on subsequent reproductive performance and milk production in high-producing dairy cows under heat stress conditions. In experiment I, addressing the pharmacokinetics of melatonin treatment in lactating dairy cows, doses of untreated, 3, 6, 9 or 12 implants/animal (18-mg melatonin each implant) were given as subcutaneous implants on gestation day 120–20 multiparous lactating dairy cows (four cows/dose group). Experiment II was performed during the warm season on 25 heifers and 114 high milk-producing Holstein-Friesian cows. Animals were randomly assigned to a control (C) or melatonin group (M). Animals in the M group received nine implants (heifers) or 12 (cows) of melatonin on day 220 of gestation. In experiment I, cows in the 12 implants group showed a higher maximum melatonin concentration (C_max) and area under the concentration curve from treatment day 0 to day 49 (AUC_0–49d) than those in the remaining groups, among which there were no significant differences in this variable. In experiment II, the likelihood of repeat breeding syndrome (<3 vs ≥4 AIs per cow) and pregnancy loss (first trimester) were 0.36 and 0.19 times lower in treated than control animals, respectively. Plasma prolactin levels decreased significantly (p = 0.01) after melatonin treatment and recovered during the postpartum compared to control cows. No significant effects on milk production were observed in the subsequent lactation. Significant differences in days open between groups (means 123 ± 71.9 and 103 ± 43, respectively, for C and M; p = 0.02) were registered. In conclusion, melatonin treatment in the early dry-off period improves the reproductive performance of dairy cattle, reducing the number of days open, repeat breeding syndrome and pregnancy loss.

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.

Puberty is the result of a dynamic interaction between genetic factors and environmental cues, all of which lead to the attainment of reproductive capacity. Thus, significant changes in hormone secretion occur from the pre-pubertal to the pubertal stage. The objective of this review is to provide an update of some endocrine, physiological, metabolic and genetic concepts involved in the establishment of the hypothalamic-hypophyseal-gonadal axis function promoting the onset of the reproductive function during puberty. To achieve this purpose, basic aspects of the function of the hypothalamic-hypophyseal-gonadal axis, the control of the axis by neurotransmitters and the interaction between reproductive function and metabolic status will be considered. Finally, the role of the novel kisspeptin system and the GPR54 receptor as modulators of puberty will be considered, in addition to the hierarchical expression of the main genes acting as regulators of the onset of puberty.

It was the aim of this field study to evaluate two different protocols of ovulation synchronization for the treatment of ovarian cysts and their effect on reproductive performance in dairy cows. In addition, factors with a possible influence on treatment success and pregnancy outcome as well as costs per pregnancy were analysed. The study was performed with 130 German Holsteins with ovarian cysts diagnosed on days 55 to 60 postpartum. Cows belonging to group 1 (n = 65) received a modified ovsynch protocol [day 0: 0.15 mg cloprostenol (PGF) + 0.02 mg buserelin acetate (GnRH); day 14: PGF; day 16: GnRH]. Group 2 (n = 65) was treated with the conventional ovsynch protocol (day 0: GnRH; day 7: PGF; day 9: GnRH). Timed artificial insemination was performed 20 to 24 h later. Cows without ovarian cysts served as controls. Treatment success (disappearance of the ovarian cyst) after the first ovsynch cycle was higher in group 1 (66.2%) than in group 2 (23.1%, p < 0.05). Reproductive measures in group 1 were comparable with those of the control group and, compared with group 2, were conspicuously better (66.2%, 76.9%, 83.1%, 59.5% vs. 40.0%, 50.7%, 60.0%, 27.5% for cumulative pregnancy rate after treatment cycle 1 to 3 and second service conception rate, respectively, p < 0.05). Overconditioned cows and cows with larger ovarian cysts showed a diminished treatment and pregnancy success. In group 1, costs per pregnancy were only slightly higher than in the control group (group 1: €352.44, group 2: €484.59, control group: €333.77). In conclusion, our results suggest that ovsynch protocols can be used in the treatment of ovarian cysts. The modified ovsynch protocol led to a better cure rate as well as a better reproductive performance, and was economically beneficial compared with a conventional ovsynch protocol.

The corpus luteum (CL) undergoes regression by prostaglandin (PG)F_2α from uterus and endothelin-1 (ET-1) plays an important role during luteolysis as a local mediator of PGF_2α in the cow. Endothelial cells (EC) and luteal cells are main cell types making up the CL and their interactions are vital for CL function. We aimed to examine the relevance of interactions between EC and luteal cells on stimulation of genes which involved ET-1 synthesis by PGF_2α. We further focused the impact of maturity of luteal cells on the stimulation of the genes. To make a microenvironment which resembles the CL, we used bovine aortic endothelial cells (BAEC) and luteinizing or fully-luteinized granulosa cells (GC) and evaluated the effect of PGF_2α on the expression for mRNA of ET-1 system by using real-time RT-PCR. PGF_2α stimulated the expression of preproET-1 and endothelin converting enzyme-1 mRNA only in the co-cultures of BAEC with fully-luteinized GC, but not with luteinizing GC. The data suggest that interactions between BAEC and fully-luteinized GC enhance the capability of BAEC to produce ET-1 in response to PGF_2α. This mechanism may contribute to the local induction of luteolytic action of PGF_2α which is dependent on the age/maturation of the CL.

The purpose of this study was to examine the effect of time for mating and gestation length on reproductive efficiency in dogs. Groups of eight, six and six beagle bitches were mated with a total of three sires on days 3, 5 and 7, respectively, after the luteinizing hormone (LH) surge. All the bitches whelped successfully. The gestation lengths (the intervals from the LH surge to the whelping) were 65.1 ± 1.9, 65.5 ± 1.9 and 68.0 ± 1.8 days, respectively. This length of mating 7 days after the LH surge was significantly longer than that of mating 3 and 5 days after the LH surge (p < 0.05). The litter sizes were 5.5 ± 2.2, 6.2 ± 1.9 and 4.8 ± 2.1, respectively and there was no significant difference between any of the groups. In conclusion, mating 3–7 days after a bitch's LH surge results in a good reproductive efficiency and the intervals from the LH surge to the whelping of mating 7 days after the LH surge shows a longer day significantly in comparison with mating 3 and 5 days after the LH surge.

The fertility of ram spermatozoa that had undergone flow cytometric sorting (MoFlo® SX) and cryopreservation was assessed after low-dose insemination of synchronized Merino ewes. Oestrus was synchronized with progestagen-impregnated pessaries, PMSG and GnRH treatment. Ewes (n = 360) were inseminated with 1 × 10⁶, 5 × 10⁶ or 15 × 10⁶ motile sorted frozen-thawed (S₁, S₅, or S₁₅ respectively) or non-sorted frozen-thawed (C₁, C₅ or C₁₅ respectively) spermatozoa from three rams. An additional group of ewes were inseminated with 50 × 10⁶ motile non-sorted frozen-thawed spermatozoa (C₅₀) to provide a commercial dose control. The percentage of ewes lambing after insemination was similar for C₅₀ (24/38, 63.2%), C₁₅ (37/54, 68.5%), S₁₅ (38/57, 66.7%), S₅ (37/56, 66.1%) and S₁ (32/52, 61.5%) groups (p > 0.05), but lower for C₅ (19/48, 39.6%) and C₁ (19/55, 34.5%) treatments (p < 0.05). This study demonstrates sorted ram spermatozoa are equally fertile to non-sorted spermatozoa even when inseminated at 2% of the dose. Furthermore, at very low artificial insemination doses (1 or 5 million motile) the fertility of sorted ram spermatozoa is superior to non-sorted spermatozoa inseminated in equal numbers. These results have significance for the future commercialization of sex-preselection technology in sheep as a reduction in the minimum effective sperm number will allow a corresponding decrease in the associated cost per dose.

In this study we have examined luteal function in non-lactating and late lactation dairy cows on day 5 of the cycle, during the period of the post-ovulatory progesterone rise. Comparison of luteal progesterone content and in vitro synthetic capacity with circulating plasma progesterone demonstrated that circulating progesterone concentration is a function of total luteal activity rather than the activity of individual units of tissue. Incubation of luteal tissue in vitro demonstrated stimulatory activity of LH and IGF-I, and to a greater degree IGF-II, on luteal progesterone synthesis. Finally the study showed no effect of double ovulation on luteal function. Occurrence of double ovulation in 35% of animals was not associated with any difference in luteal function or plasma progesterone concentrations.

Over a 25-month period 8118 blood samples were assayed for the presence of the serum pregnancy specific-protein B (PSPB) and progesteron (P4) concentrations on three Hungarian large-scale dairy farms. Pregnancy (n = 4085) was checked by BioPRYN assay at 30–36 days post-insemination (PI). Samples from all cows that tested not pregnant and from cows with an optical density (OD) reading in the BioPRYN test that was between 0% and 30% above the cutoff OD value were tested for serum P4 concentration. According to serum P4 concentration, cows were assigned to three categories: high (>4 ng/ml), medium (2–4 ng/ml) and low (<2 ng/ml) serum progesterone. The authors predicted a presumed (low) or possible (medium) late embryonic loss (LEL) or maintenance of the pregnancy (high). A total of 710 LELs were detected (17.4%) and 31.8% of them were predicted because of a low OD value at 30–36 days after insemination. Lower PSPB serum level significantly refers for LEL (p < 0.0001). The prediction rate for the true embryonic loss was 31.8% when OD cutoff from 0% to + 30% of cutoff was examined while it was 62.5% when the threshold was OD cutoff of 0% to 10% of cutoff. The authors conclude that BioPRYN was useful for prediction of a part of LEL in dairy cows and serum P4 concentration in these cows related to the rate of LEL.

A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 × 10⁶), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35–40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5°C and 5% CO₂ in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 μl droplets of CCM were incubated for 1 h. Afterwards, 5 μl of CCM containing 25 000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.

The aim of the present study was to assess the effects of the ovum pick-up (OPU) technique on animal well-being. Eight dairy heifers were subjected to 4 months of twice-weekly OPU. The physiological response to OPU was recorded in four heifers at two sessions, at the beginning (time 1) and at the end (time 2) of the 4-month period. Heart rates were measured and blood was analysed for cortisol, vasopressin and PG-metabolite before, during (every 5 and 2 min), and after the OPU sessions. Reactions to each subprocedure of OPU (‘restraint’, ‘epidural’, ‘device in’ and ‘puncture’) were closely observed. In all heifers, reactions to the OPU procedures were also noted throughout the experimental period, and changes in routine behaviour, oestrous behaviour, body temperature, or other clinical traits were recorded. Subsequent to the experiment, the ovaries and tails were carefully inspected. At time 1, there was an insignificant increase in heart rate and cortisol throughout the OPU procedure. At time 2, these two parameters increased significantly, but both parameters declined to pre-OPU levels 10 min after completion of the procedure. No significant changes were seen in vasopressin or PG-metabolite at time 1 and time 2. Behaviourally, the heifers showed the strongest response to epidural anaesthesia, with a tendency for more intense response during the late 4-month sessions. The response to ‘device in’ and ‘puncture’ varied among individuals independently of time. There were no changes in the routine or oestrous behaviour throughout the experiment and no signs of clinical disorders. No major pathological changes were macroscopically seen in the ovaries and tails subsequent to the 4 months of OPU. In conclusion, the heifers showed a response to OPU, mostly to administration of epidural anaesthesia. However, we demonstrated that epidural anaesthesia can be administered in a way causing less discomfort.

The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6–8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 × 10⁶ motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 × 10⁶ motile spermatozoa. The sows were anesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero-tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 × 10⁶ motile spermatozoa. The sows were anesthetized 61.1 ± 12 h after insemination (48–72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI-group, and from unilateral side of the reproductive tract in the DIUI-group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI- than the DIUI-group (p < 0.001). In experiment II, fertilization occurred in five of eight sows (62.5%) after DIUI. The number of ova that ovulated were 16.4 ± 2.6 per sow and the embryos numbering 11.4 ± 2.3 per sow were recovered from both sides of the reproductive tract. In conclusion, the spermatozoa given by DIUI could be recovered from only one side of the reproductive tract of sows at approximately 24 h after DIUI via the flushing technique. However, embryos were found in both sides of the oviducts and the proximal part of the uterine horns 48–72 h after insemination, indicating that the fertilization occurred in both sides of the oviducts.

During physiological pregnancy, all tissues and, mostly, placenta and foetus require high amounts of oxygen. Reactive oxygen species (ROS), generated both by mother and foetus, are implicated in foetal growth because they promote replication, differentiation and maturation of cells and organs. Nevertheless, ROS excess, if not properly counterbalanced, may lead to an alteration in cell constituents, with harmful effects both on mother and foetus.ROS exert a biphasic effect because adequate ROS concentration is essential for embryo development, implant, foetal defence against uterine infections, steroidogenesis, pregnancy maintainance and partum. On the other hand, an uncontrolled ROS generation, beyond physiological antioxidant defences, may lead to embryo resorption, placental degeneration with subsequent alteration in maternal-foetal exchanges, delay in foetal growth, pregnancy interruption, stillbirths. This review investigates the mechanisms underlying ROS generation and effects, throughout physiological and pathological pregnancy in sheep, with a look to antioxidants and their importance in such a critical phase of the reproductive cycle of the sheep.

Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow-derived MSCs (BM-MSCs) at molecular and cellular level. Buffalo BM-MSCs were isolated by Ficoll density gradient method and cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony-forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM-MSCs have the capacity to form plastic adherent clusters of fibroblast-like cells and were successfully maintained up to 16^th passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT-PCR analysis and protein localization study showed that buffalo BM-MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM-MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM-MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy.

This study was conducted to explore the influencing factors of ova in vitro fertilization (IVF) and transfer of the fertilized ova into the oviduct of recipient hens. The efficiency of fertilization was compared using three aspects: (i) the different time of ova collection and transfer, (ii) egg-laying period of recipient hen; and (iii) semen volume. The following results are observed: 72%, 40% and 0% of ova were found in ovarian sac in 30∼40 min, 50∼60 min and more than 90 min post-oviposition, respectively; 20%, 18%, 14% and 5.8% of ova were fertilized with 0.1, 0.2, 0.5 and 1.0 ml semen, respectively; and 33% and 100% of healthy chickens were hatched from fertile ova with 0.1 and 0.5 ml of semen, respectively. All oocytes obtained from ovary and mid-oviduct were unfertilized. Embryos were transferred into recipient hens 30 min ± 10 min post-oviposition, and 70% of shelled eggs were produced. There were no eggs produced in the other transfer times. This demonstrated that live chicken can be obtained by IVF of ova collected shortly after oviposition. It was important that the ovum was transferred into the oviduct infundibulum of recipient hens immediately or shortly after oviposition.

The proportion of type A spermatogonia in the isolated testis cells is a prerequisite for conducting experiments and the manipulation of these germ cells. Thus, this study was designed to examine the wide range of strategies for the isolation, identification and enrichment of type A spermatogonia in pre-pubertal buffalo calves (3–6 months). Histological findings revealed the presence of maximum number of type A spermatogonia at 5 months, which was further confirmed by DBA immunohistochemistry. In a newly modified strategy for the isolation of testis tissues, mincing followed by trituration and two rounds of digestion with collagenase, hyaluronidase and DNase yielded more than 95% testis cell population. Differential plating with laminin, poly-l-lysine and gelatin significantly (p < 0.05) affected the purity of type A spermatogonia. Among these extracellular matrix (ECMs) molecules, laminin and gelatin performed well and reached at a purity of 39.38 ± 1.21% and 32.15 ± 1.60%, respectively. In addition, combination of laminin and gelatin followed by Percoll centrifugation performed the best and yielded >90% type A spermatogonial purity. Moreover, viability of the cells was not affected (p > 0.05) irrespective of different enrichment methods. In conclusion, type A spermatogonia isolation and enrichment system was developed using different ECM molecules in buffaloes, which will aid in solving wide range of problems especially fertility-related problems and transgenic animal production in buffaloes.

The study was designed to examine the effects of calcitonin (CT) on the development of murine pre-implantation embryos and possible molecular mechanisms involved in the process. In the present study, the 2-cell embryos were treated with different concentration of CT in vitro for the indicated time and the results demonstrated that CT promoted the development of the pre-implantation embryos in a dosage-dependent manner by increasing the intracellular Ca²⁺ level. Furthermore, the present study showed that CT significantly increased the expression of phospho-P38MAPK (Mitogen-Activated Protein Kinase) of the pre-implantation embryos by Western blots and pre-treatment of specific P38MAPK inhibitor significantly reduced the promotion effects of CT on the embryonic development in vitro culture. Moreover, the results of intrauterine horn injection showed that the average number of embryos implanted in CT-antibody or specific P38 MAPK inhibitor-treated uterus was significantly lower than that of the corresponding control, respectively. And the observation of tissue specimen suggested that some embryos were degenerated in CT-antibody or specific P38 MAPK inhibitor-treated uterus, and adipose vacuoles were present in the decidual cells. In conclusion, CT promoted the development of pre-implantation embryos and the intracellular Ca²⁺-dependent P38MAPK signal molecule was involved in the process.

This study investigated the effect of altrenogest treatment on the farrowing development of sows, and birth weight (BW) and piglet survival until the third day of life. Three control groups were used: (i) sows that farrowed spontaneously before 114 day of gestation (CONT <114); (ii) sows that spontaneously farrowed at ≥114 day of gestation (CONT ≥114); (iii) sows that farrowed at ≥114 day with cloprostenol treatment (CONTCLOPR). Other sows were treated with altrenogest (Regumate^®) for 3 days (days 111, 112 and 113 of gestation): one group gave birth spontaneously (ALT) and the other group received altrenogest until day 113 and cloprostenol on day 114 (ALTCLOPR). There were no differences (p > 0.05) in farrowing duration, BW, coefficient of variation (CV) of BW, stillborn piglets, mummified foetuses, percentage of light piglets and survival until Day 3 between sows with and without cloprostenol treatment, in both control (CONT ≥114 vs CONTCLOPR) and altrenogest-treated sows (ALT vs ALTCLOPR). Further comparisons were performed taking into account three groups: sows with early delivery (CONT <114 – farrowing before 114 days of gestation; n = 56), sows with longer gestation (CONT ≥114 – with and without cloprostenol treatment sows; n = 103) and ALT sows (with and without cloprostenol treatment; n = 105). Gestation length of CONT ≥114 and ALT sows was similar (p > 0.05), but higher than in CONT <114 sows. There were no differences (p > 0.05) between groups in farrowing duration, CV of BW, and percentages of stillborn piglets and mummified foetuses. Sows of CONT <114 group had a larger litter size and a lower BW than sows of the other two groups (p < 0.05). Sows of CONT <114 group had a higher percentage of lighter piglets and a lower piglet survival rate (p < 0.05) than ALT sows. In conclusion, altrenogest treatment proved to be an efficient method to avoid early parturition in 3–5 parity sows resulting in heavier piglets at birth.

The growth factor receptor-bound protein 14 (Grb14) is a cellular adapter protein belonging to the Grb7 family of proteins. Studies with human and rodent cells have demonstrated that Grb14 acts as a negative regulator of tyrosine kinase receptor signalling through the MAPK and PI3K pathways. In cattle, tyrosine kinase receptors are activated during follicular development but the role of Grb14 in this process has not yet been investigated. Therefore, the aim of the present study was to characterize Grb14 mRNA expression in ovarian somatic cells during follicular growth and deviation in cattle. We found Grb14 mRNA expressed in both granulosa and theca cells derived from follicles at different stages of development (3–5 , 6–8, >8 mm in diameter). The abundance of mRNA for Grb14 was higher in granulosa cells of subordinate compared with those from dominant follicles at days 3 and 4 of the follicular wave (p < 0.05). Further, there was a negative correlation between the abundance of mRNA for Grb14 and P450Arom in granulosa cells (R² = 0.367; p < 0.05) and between the abundance of mRNA for Grb14 in granulosa cells and concentration of oestradiol in follicular fluid (R² = 0.545; p < 0.05). In theca cells, the expression of Grb14 mRNA did not differ between dominant and subordinate follicles (p > 0.05). These findings suggest that Grb14 may play a regulatory role in granulosa cells during follicular deviation in cattle.

The aim of this work was to study the influence of embryonic and maternal genotype of two lines of rabbits selected by growth rate (line R) and litter size at weaning (line A) on prenatal survival. Embryos were recovered at 48 h of gestation from R and A donors (39 and 35 does, respectively) and reciprocally transferred to the oviducts of recipient does to the R (n = 15) and A (n = 14) lines. Each recipient doe received six embryos from line R into one oviduct and six embryos from line A into the other. Recipient does were examined by laparoscopy to determine implantation rate on day 14 and slaughtered on day 25 of gestation to determine the number of live foetuses and the weight of foetuses and placentas. No differences were found between lines in fertilization rate and stage of embryo development at 48 h post-insemination. Implantation rate was affected by both the embryonic and maternal genotype. While embryos from donor line A had the highest implantation rate (0.78 ± 0.032 vs 0.65 ± 0.036 for line R), recipient line R had a better implantation rate (0.78 ± 0.033 vs 0.64 ± 0.036 for line A). Foetal survival was affected by the embryonic genotype. Embryos from donor line A had a higher foetal survival rate than embryos from donor line R (0.65 ± 0.036 vs 0.53 ± 0.038, respectively) but lower foetal and placenta weights. In conclusion, while embryonic genotype influenced both implantation and foetal survival rate, R embryos had the lowest rates, maternal genotype affected the implantation rate and R recipients may show a greater uterine receptivity during implantation period. Moreover, it must be observed that foetal and placenta weights were significantly affected by embryonic genotype and heavier for R line.

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post-thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris-citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm) or trehalose (100 mm), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT-154 (anti-phosphotyrosine antibody) and FITC-conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post-thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.

Plasmatic concentrations of von Willebrand Factor (vWF) increase during pregnancy in humans and dogs; however the mechanism of such increase is still not well defined. The aims of this study were: (i) to evaluate changes in vWF concentration during pregnancy and during the subsequent oestrous cycle in bitches affected and unaffected by von Willebrand Disease (vWD); (ii) to correlate the vWF levels and cortisol levels in both groups. Seven vWD affected (GI) and nine unaffected (GII) bitches were used. The animals were assessed during pregnancy, parturition, lactation and non-gestational oestrous cycle in 11 moments (Pregnancy 1, Pregnancy 2, Parturition, Lactation 1, Lactation 2, Lactation 3, Anestrus, Proestrus, Oestrus, Diestrus 1, and Diestrus 2). The following tests were performed; measurement of von Willebrand factor antigen (vWF:Ag), albumin and cortisol. In both groups, vWF concentration remained stable during the non-gestational oestrous cycle, but increased during pregnancy, with the highest value observed at parturition. Increases of 70% and 124% in vWF were seen in GI and GII, respectively, compared to anestrus. No correlation was found between vWF and cortisol. Values of vWF:Ag changed during pregnancy, with a peak at parturition, both in vWD affected and unaffected animals. Values of vWF were not altered in the different phases of the oestrous cycle following pregnancy in both groups. Evaluation of vWF during pregnancy can cause false negative results for vWD, but assessment can be performed at any point in the oestrous cycle of non-pregnant bitches.

The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin β_B subunits were not detected, and inhibin/activin β_A subunit was detected, in the luminal and glandular epithelium on cyclic day 13. Real-time polymerase chain reaction and Western blotting results for the inhibin/activin subunits suggested a significant increase in the expression of inhibin/activin subunit β_B and a significant decrease in the expression of inhibin/activin subunit β_A on pregnancy day 25 compared with those on cyclic day 13. Enzyme-linked immunosorbent assays suggested a significant decrease in the concentration of activin A in endometrium extracts from cyclic day 13 to pregnancy day 25. These results suggest that inhibins or activins synthesized in the uterus, as endocrine factors and necessary nutriments, have different expression patterns and may play different, important roles during early embryonic development of the equine.

We examined the effect of prolonged high heat stress on reproductive performance and its relationship with gene expression in pre-implantation embryos and endometrial tissue. In experiment 1, primiparous rabbit does were divided into two environments: control does (maintained between 14 and 22°C) and heat-treated does housed in a climatic chamber (maintained between 25 and 35°C). Females were reproducing, and the litter size and live born kits were assessed at 2nd and 3rd partum. In heat-treated does, lower litter size (9.7 ± 0.48 and 11.4 ± 0.50) and fewer live born kits (7.2 ± 0.55 and 10.2 ± 0.57) were observed, although similar ovulation rates and numbers of pre-implantation embryos were noted. In experiment 2, after 3rd partum multiparous non-lactating does from each experimental group were used to obtain pre-implantation embryos and endometrial tissue. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-ɣ, HSP70 and HSP90 were analysed by real-time qPCR. Higher values of OCT-4 expression were observed in embryos and endometrial tissue in females reproduced under heat conditions. Moreover, elevated temperatures have been shown to up-regulate VEGF in embryos and down-regulate Ifn-ɣ in endometrial tissue. The findings suggest a deleterious temperature effect on litter size and live born kits as a consequence of variation in gene expression pattern of the pre-implantational embryo and the endometrium associated with proliferation and differentiation and probably with implantation and uterine and foetal development during gestation.

To achieve optimal reproductive performance in pig herds, sows need to become pregnant as soon as possible after weaning. The aim of this study was to investigate herd and management factors associated with reproductive performance of sows after weaning. A questionnaire pertaining to sow management at weaning and herd reproductive data were collected from 76 randomly selected commercial pig herds in Belgium. Associations between the herd factors and two reproductive parameters after weaning (weaning-to-oestrous interval: WEI and percentage of repeat breeders: RB) were analysed using general linear mixed models. A separated feeding strategy of breeding gilts from 60 kg onwards was significantly associated with a shorter WEI (5.54 vs 7.28 days; p = 0.040). Factors significantly associated with a lower percentage of RB were housing the newly weaned sows separated from the gestating sows (7% vs 12%; p = 0.003), using semen < 4 days after collection (7–9 vs 14%; p = 0.014) and stimulating oestrus twice a day (8 vs 11%; p = 0.025). In conclusion, some management practices, such as feeding strategy of breeding gilts, housing conditions of sows, method of oestrous stimulation and storage duration of semen, have an influence on the outcome of reproductive parameters such as weaning-to-oestrous interval and percentage of repeat breeders. These practices can be implemented rather easily by pig producers and may consequently lead to improvements of reproductive performance of sows after weaning.

The objective of this study was to assess the effects of Coxiella burnetii shedding or seropositivity on post-partum recovery and subsequent fertility in high-producing dairy cows. Given the difficulty in diagnosing C. burnetii infection at the farm level, an exhaustive series of tests in 43 pregnant animals that delivered at least one live calf were conducted, including blood serology and PCR of milk or colostrum, cotyledons (only at parturition), faeces, vaginal fluid against C. burnetii on gestation Day 171–177, at parturition and on Days 1–7, 8–14, 15–21, 22–28, 29–35 and 90–97 post-partum. During scheduled herd visits, ultrasonography (US) of the genital tract and examination of vaginal fluid were performed on Days 15–21 (V1), 22–28 (V2), 29–35 (V3) and 51–57 (V4) post-partum by the same veterinarian. Logistic regression analysis revealed that the likelihood of suffering endometritis (the presence of echogenic intrauterine fluid (IUF), cervical diameter of ≥4 cm or endometrial thickness ≥0.75 cm) was lower in C. burnetii-seropositive animals (OR = 0.10), compared with C. burnetii-seronegative animals. According to Kaplan–Meier survival analysis, C. burnetii-seronegative and non-shedding cows showed a delayed return to luteal activity and conception was delayed in non-shedding animals, compared with the remaining animals. Overall, the results of our study provide useful insight into the effects of C. burnetii infection on post-partum recovery and subsequent fertility. In particular, animals not infected with Coxiella seem to be susceptible to infection and not protected against the bacterium in dairy herds. The elevated costs of determining an infection at the farm level, make monitoring of cows virtually impossible from a clinical point of view.

Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14⁄PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin⁄PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = −0.41) and with plasma membrane integrity (p = 0.01; r = −0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.

Androgens are one of the most important agents influencing ovarian follicles growth and development. The biological action of androgens is primarily exerted through transcriptional regulation by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The purpose of this study was to test the role of androgen receptor agonist testosterone (T) or antagonist 2-hydroxyflutamide (2-Hf) and in combination on AR expression in cultured porcine granulosa cells (GC) or whole follicles. Granulosa cells isolated from mature pig follicles were cultured for 48 h. During the last 12 and 24 h of culture, they were incubated in the presence of T (10⁻⁷ m/ml), 2-Hf (1.7 × 10⁻⁴ m) or both T and 2-Hf (T + 2-Hf, at the same concentrations as when added separately). To better imitate in vivo conditions, whole follicles (6–8 mm in diameter) isolated from porcine ovaries have been incubated (for 12 and 24 h) in an organ culture system with the addition of the same factors. Thereafter, cells or sections obtained from cultured follicles were processed for AR detection by immunocytochemistry or immunohistochemistry. Moreover, expression of AR mRNA and protein was determined by real-time PCR and Western blot analysis. It was shown that the addition of 2-Hf in the presence of T had a positive effect on AR mRNA and protein expression in porcine GC and ovarian follicles. Moreover, the addition of 2-Hf influenced AR distribution in GC cultures which is seen as change of its localization from nuclear to perinuclear. Our results suggest that androgens acting through AR could be involved in the control of AR expression in porcine GC in vitro and in vivo.

The aim of this study was to evaluate the effects of different treatments for induction and synchronization of oestrus and ovulation in seasonally anovulatory mares. Fifteen mares formed the control group (C), while 26 mares were randomly assigned to three treatment groups. Group T1 (n = 11) were treated with oral altrenogest (0.044 mg/kg; Regumate^®) during 11 days. Group T2 (n = 7) was intravaginally treated with 1.38 g of progesterone (CIDR^®) for 11 days. In group T3 (n = 8), mares were also treated with CIDR^®, but only for 8 days. All mares received PGF2α 1 day after finishing the treatment. Sonographic evaluation of follicles, pre-ovulatory follicle size and ovulation time was recorded. Progesterone and leptin levels were analysed. Results show that pre-ovulatory follicles were developed after the treatment in 88.5% of mares. However, the pre-ovulatory follicle growth was dispersal, and sometimes it was detected when treatment was not finished. While in mares treated with intravaginal device, the follicle was soon detected (1.5 ± 1.2 days and 2.3 ± 2.0 days in T2 and T3 groups, respectively), in T1 group, the pre-ovulatory follicle was detected slightly later (3.9 ± 1.6 days). The interval from the end of treatment to ovulation did not show significant differences between groups (T1 = 13.1 ± 2.5 days; T2 = 11.0 ± 3.6 days; T3 = 13.8 ± 4.3 days). The pregnancy rate was 47.4%, similar to the rate observed in group C (46.7%; p > 0.05). Initial leptin concentrations were significantly higher in mares, which restart their ovarian activity after treatments, suggesting a role in the reproduction mechanisms in mares. It could be concluded that the used treatments may be effective for oestrous induction in mares during the late phase of the seasonally anovulatory period. Furthermore, they cannot synchronize oestrus, and then, it is necessary to know the reproductive status of mares when these treatments are used for oestrous synchronization.

The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(−) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes.

Quantitative reverse transcription PCR (RT-qPCR) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre-implantation embryos. However, the high sensitivity of this technique requires that to prevent bias in expression data interpretation, the reference genes used for normalization are fully validated before use. Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte. In the present study, we evaluated eight commonly used reference genes such as ACTB, GAPDH, H2AFZ, HPRT1, PPIA, SDHA, TUBB and YWHAZ and for sheep oocyte RT-qPCR before and after in vitro maturation. We have also compared different cDNA priming strategies using random hexamers or oligo-dT. GeNorm analysis of the results identified the most reliable genes for normalization to be SDHA, TUBB and PPIA when oocyte cDNA was made with random hexamers, and YWHAZ, TUBB and SDHA when oligo-dT primers were used (H2AFZ and HPRT1 were excluded from the geNorm analysis). Interestingly, the analysis revealed that the least stable genes were ACTB and GAPDH, which are the conventional ‘housekeeping’ genes used in many studies. We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the cDNA priming strategy employed.

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post-partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post-partum patterns of IGF-1, insulin, β-hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid-related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post-partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post-partum dairy cows.

The successful outcome of an insemination is a combination of both male and female fertility-linked factors. We investigated the first service conception rate of cows at artificial insemination (AI) in the smallholder dairy farms in Bangladesh. Frozen straws were prepared from ejaculates of Bos indicus (n = 7) and Bos indicus × Bos taurus (n = 7) AI bulls. Fertility was determined from 6101 first services in cows that were performed by 18 technicians in four regions between April 2004 and March 2005. Pregnancy was diagnosed by rectal palpation between 60 and 90 days post-insemination. The Asian version of Artificial Insemination Database Application (AIDA ASIA) was used for bulls-, cows- and AI-related data recording, and later retrieved for analysis. The mean ± SD number of inseminations performed from individual bulls and their conception rates were 436.0 ± 21.6 and 50.7 ± 1.9%, respectively. Logistic regression demonstrated body condition scores (BCS), heat detection signs, months of AI and their interactions had greatest effects (odds ratios: 1.24–16.65, p < 0.04–0.001) on first service conception rate in cows. Fertility differed (p < 0.02–0.001) between the regions, previous calving months, months of AI, BCS, parity and heat detection signs of cows. Inseminations based on mounting activity (n = 2352), genital discharge (n = 3263) and restlessness and/or other signs (n = 486) yielded a conception rate of 53.6%, 48.8% and 50.1%, respectively (p < 0.05). Conception rate between technicians ranged between 43.4% and 58.6% (p < 0.05). The days interval from calving to first service (overall mean ± SD = 153.4 ± 80.6) had relationship (p < 0.001) with BCS, months of previous calving and parity of the cows. Fertility at AI in smallholder farms can be improved by training farmers on nutrition and reproductive management of the cows.

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris-based extender on the post-thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris-fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris-egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris-based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris-based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.

The present study investigated the effects of pre-weaning energy substitutions on follicular development, endocrine characteristics and subsequent litter size in primiparous sows. Sows were fed a standard lactation diet (14.1 DE MJ/kg) and then allocated to a Control (C, n = 24), Fat (F, n = 23), Sugar (S, n = 23) or post-weaning Regumate (positive control; R, n = 22) treatment at 9 days before weaning of the C, F and S treatments. During the treatment period (8 days), 1 kg of the lactation diet was substituted with 1 kg of a fat-rich (F, 23.85 DE MJ/kg) or sugar-rich (S, 15.75 DE MJ/kg) substitution for F and S sows, respectively. For the R treatment, sows were weaned 8 days earlier than other treatments and fed a lactation diet at 3.5 kg with two doses of altrenogest as topdressing from 1 day before weaning until the day on which the other sows were weaned. The F treatment aimed to increase energy intake, and the S treatment aimed to elevate post-prandial glucose and insulin concentrations. Weaning-to-ovulation interval tended to be reduced in the S treatment compared with C (p = 0.06) and F (p = 0.08) treatments. Body weight (BW) loss during the treatment period, post-weaning follicle development, plasma oestradiol and pre-weaning leptin did not differ among C, F and S sows, although BW loss was lower and leptin was higher in the R treatment. Post-ovulatory progesterone concentration in the S treatment was higher (p < 0.05). Sows in the S and R treatments had a greater proportion of litters with larger litter sizes (p < 0.05). The outcome suggests that increasing circulating insulin and glucose concentrations during late lactation or a week of metabolic recovery positively improves subsequent litter size in primiparous sows.

The aim of this study was to evaluate the possible association between hormonal changes that occur during oestrus and biomarkers related with glucose metabolism (glucose and insulin), lipid metabolism (lipidic profile and BChE) and adipokines (adiponectin and ghrelin) in healthy bitches. For this purpose, we measured these analytes in serum of bitches, at two times: before (T1) and after (T2) the LH peak that were established according to progesterone concentrations. Increased levels of total cholesterol (p < 0.01), high density lipoprotein cholesterol (HDL-C) (p < 0.01), low density lipoprotein cholesterol (LDL-C) (p < 0.01), adiponectin (p < 0.01) and ghrelin (p < 0.05) were observed at T2 in comparison with T1. No statistically significant changes were observed in serum glucose, insulin, homoeostasis model assessment for insulin sensitivity (HOMA), triglycerides and BChE. When all data of T1 and T2 were pooled, serum adiponectin showed positive correlation with progesterone (r = 0.353; p = 0.022) and HDL-C (r = 0.307; p = 0.048), and negative with insulin (r = −0.429; p = 0.005), HOMA (r = −0.446; p = 0.003) and BChE (r = −0.522; p < 0.001). Ghrelin showed negative correlation with estradiol (r = −0.701; p = 0.004). BChE was negatively correlated with estradiol (r = −0.441; p = 0.018) and glucose (r = −0.343; p = 0.028), and positively with insulin (r = 0.460; p = 0.003) and HOMA (r = 0.505; p < 0.001). In conclusion, changes in metabolic biomarkers occur in bitches after LH peak, characterized by increased lipids (total cholesterol, HDL cholesterol and LDL cholesterol) without changes in BChE activity, and increased adiponectin and ghrelin concentrations, without significant changes in glucose and insulin.

The bacterial load and degree of antibiotic resistance present in untreated and antibiotic-treated semen samples were investigated in five bulls standing at a cattle-breeding centre. Bacterial load was determined by colony counts from semen samples cultured on brain heart infusion and nutrient agar plates. Antibiotic resistance in these bacteria was assessed by measuring the diameter of bacterial growth inhibition zones around discs containing different concentrations of antibiotics. Representative antibiotic-resistant bacterial isolates were selected for identification. Untreated semen contained few culturable bacteria, and all were completely sensitive to gentamycin, spectinomycin and lincomycin: six of the isolates showed some resistance to tylosin. In semen to which antibiotics had been added as part of the routine production process, two isolates were sensitive to all of the antibiotics tested, and the remainder were resistant to all. Resistant Gram-negative isolates that were identified included Pseudomonas and Stenotrophomonas spp. both in the class Gammaproteobacteria and a Sphingomonas sp. which is in the class Alphaproteobacteria.

Here is reported a disorder of sex development found in the Portuguese Lusitano horse breed. The complex genital phenotype included mammary glands, abdominal testes without epididymis, connected through oviducts to pelvic hypoplastic uterine horns and fused vulvar labia majora from which protruded ventrally a penis-like structure. This structure was presented in a reversed position, the urethral opening placed dorsally in the glans. However, it was functional both for micturition and erection. The horse exhibited female micturition posture and aggressive male-like behaviour, including flehmen, mounting, thrusting and flagging of the tail. Plasma testosterone concentrations were below detection limits and the genetic evaluation revealed a 64, XX, SRY-negative karyotype. Surgery consisted in the removal of abdominal gonads followed by amputation of the penis and repositioning of the urethra. This case of reversion between the chromosomal and gonadal sex, associated with mixed anatomical and behavioural phenotype, illustrates that development of the testes may occur in the absence of the SRY gene and that other genetic and cellular pathways leading to gonad differentiation should be investigated.

An anencephalic full-term porcine foetus accompanied by a mummified head was submitted for examination. The neck almost entirely lacked skin and was covered by granulation tissue as were the exposed parts of the spine and spinal cord. The case represents a rare case of intrauterine amputation. A definitive cause could not be established because the placenta was not available. The most likely cause is strangulation of the neck. Such strangulation could be due to a defect of the allantoamnion with herniation of the foetal head or entanglement by amniotic constriction bands.

Very few carnivore's embryology is reported mainly restricted to old literature without new technique analyses. Also, their development focuses on pharyngeal arches and stem cell sources and the high capacity for differentiation from those cells to generate embryonic tissue. We aimed to use immunohistochemistry to prove the potentiality of these stem cell niches. The results were to highlight the timetable for the development of dogs and cats, the proper formation of pharyngeal arches and the description of these cells on first and second arches since 17–25 days of pregnancy. After that, the differentiation process is reduced.

Previous studies indicate that reproductive prolificacy of obese swine breeds is markedly influenced by embryo losses in early pregnancy. In such period, adequate secretion of progesterone (P4) by the ovary is essential for pregnancy success. This study analyses the luteal functionality during the oestrous cycle and early pregnancy of Iberian sows and Large White x Landrace females, in terms of P4 secretion after in vitro culture of luteal tissue stimulated or not with luteinizing hormone (LH). The secretion of progesterone (expressed in ng/mg of luteal tissue or ng/mgLT) of the corpora lutea of obese Iberian swine was always hampered when compared to lean genotypes, either during early oestrous cycle (110.7 ± 37.8 vs 259.7 ± 10.2 ng/mgLT; p < 0.0001), late oestrous cycle (49.0 ± 3.5 vs 75.92 ± 7.14 ng/mgLT; p < 0.0001) or early pregnancy (38.4 ± 2.1 vs 70.7 ± 5.3 ng/mgLT; p < 0.0001). The differences in basal P4 secretion remained after stimulation with LH. Finally, P4 secretion during early pregnancy of Iberian sows decreased with age and, hence, with obesity features (46.6 ± 4.2 vs 65.5 ± 4.8 ng/mgLT; p < 0.001). In conclusion, the results of the present study provide convincing evidence of a reduced luteal function during oestrous cycle and early pregnancy of sows with obesity/leptin resistance like Iberian sows, which may contribute to the low reproductive efficiency reported in this breed.