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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The aim of this study was to evaluate powdered coconut water extender (ACP-106c; ACP Serviços Tecnológicos Ltda, ACP Biotecnologia, Fortaleza, Ceará, Brazil) as a diluent for freezing dog semen and the fertility after vaginal insemination of semen frozen therein. Ten ejaculates were collected from five dogs, evaluated fresh, diluted in ACP-106c, 10% egg yolk and 6% glycerol, cooled and frozen. In the first phase of the study, straws with frozen semen were thawed and immediately subjected to the same analysis as the fresh semen and, in addition, to Computer-Assisted Semen Analysis (CASA). In phase 2, 10 bitches that had been subjected to natural breeding during a preceding oestrous cycle were vaginally inseminated with thawed semen that had been re-diluted in ACP-106c. After thawing, a mean of 77% sperm motility was obtained through subjective analysis and 77.3% through CASA. Following artificial insemination, a 60% pregnancy rate was observed, resulting in a 50% parturition rate and a mean litter size of 3.4 (SEM 0.6), with 47.1% males and 52.9% females. ACP-106c can be successfully used for freezing canine semen, and vaginal deposition of such semen yields similar pregnancy rates to those reported in other studies.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The possibility of using cooled or frozen semen is an important tool for those working with canine breeding programmes especially as this allows the possibility of transporting semen to several countries and storing it for an indefinite period of time (Rijsselaere et al. 2005).

A powdered coconut water–based extender ACP-106c (ACP®; ACP Serviços Tecnológicos Ltda, ACP Biotecnologia, Fortaleza, Ceará, Brazil) has been developed in recent years. This medium has been used for semen freezing (Cardoso et al. 2007) and for artificial insemination (AI) with cooled semen in dogs (Uchoa et al. 2012); however, there are no reports of normal pregnancy when using ACP-106c with frozen/thawed canine semen.

After thawing, the semen is typically only subjected to standard evaluations, which primarily include motility, longevity and sperm morphology. However, the use of these parameters alone is insufficient to predict the fertilizing potential of semen. Both in vitro and in vivo analyses are of great importance in determining the extent of damage caused to the sperm cells and thus improving the methods of cryopreservation. Therefore, this study aimed to evaluate the in vitro and in vivo efficacy of powdered coconut water–based extender (ACP-106c) in freezing canine semen.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Animals

Five males (1 Beagle, 2 Boxers, 1 French Bulldog and 1 Brazilian Terrier) of proven fertility, aged 2–9 years old, and ten bitches (1 Beagle, 3 Boxers, 4 French Bulldogs and 2 Brazilian Terrier), aged 2–6 years old, originating from the Grande Canafístula Kennel, were selected for use in this study. The bitches were selected based on previous normal oestrous cycle and pregnancy. The animals were fed a commercial diet for breeding dogs, HT 42d (Royal Canin®), twice a day and given free access to water.

Experimental groups

Each female was used twice throughout the study, during consecutive oestrous cycles, for the evaluation of natural breeding (NB; Group 1) and AI with frozen semen (Group 2). Following birth from NB, each female was subjected to AI in the subsequent oestrous cycle with semen obtained from the same dog used for the NB.

Semen collection and evaluation

Two ejaculates were obtained from each male animal (n = 10) through digital manipulation, and only the sperm fraction was retained for analysis. Semen collection and freezing occurred approximately 3 months after Group 1 matings occurred. Males used in this experiment performed covers of other females during this interval, always keeping to a minimum period of 15 days without collections or matings before freezing semen collection for this study. Ejaculates were immediately evaluated for volume. Sperm concentration was determined using a Neubauer chamber. Sperm total motility and vigour were assessed under light microscopy (×100 and ×400). To assess sperm membrane function, a hypo-osmotic test was performed. A total of 100 cells were counted under light microscopy (×400) to determine the percentage of cells with swollen or coiled tails. A semen smear was produced and stained with Rose Bengal (Silva et al. 2006a) for the evaluation of sperm morphology and acrosome integrity, for which 200 cells were counted using phase-contrast microscopy (×1000). Furthermore, the percentage of live sperm cells was determined using bromophenol blue (Barbosa et al. 2009); for this assay, 100 cells were counted under light microscopy (×400), and the unstained sperm cells were classified as live cells.

Semen dilution and processing

The ACP-106c (ACP®; ACP Serviços Tecnológicos Ltda), which is used as an extender for dog semen, was used. It is composed of dehydrated coconut water, pH buffers and antibiotics (gentamicin). One package of ACP-106c weighs 18.55 g; it is reconstituted in 250 ml of distilled water, yielding a solution with an osmolarity of 355 mOsm/kg and pH of 7.22. The extender was first prepared according to the manufacturer's instructions, and then, 10% of this solution was replaced with egg yolk (ACP-yolk). Glycerol was then added to the ACP-106c-yolk extender to a final concentration of 6% (ACP-yolk-glycerol). The semen was diluted to achieve a final concentration of 200 × 106 sperm cells/ml, after the ACP-yolk-glycerol extender had been brought to a temperature of 27°C. The diluted semen was then submitted to the freezing process previously described (Mota Filho et al. 2011).

Semen thawing

Straws containing semen samples from each animal were stored for at least 7 days, and then, one straw of each ejaculate of each male was thawed in a water bath at 37°C for 1 min for semen analysis (as in the first phase of the study) and for computerized semen analysis (Phase 1). For each AI (Phase 2), eight straws (of the same ejaculate) of each male were thawed following the same experimental protocol of the Phase 1.

Phase 1: Computerized semen analysis

A computerized analysis of sperm motility was performed with the help of a phase-contrast microscope coupled to a video camera adapted to the Sperm Class Analyser® system (SCA; Microptic S.L., version 3.2.0) as previously described (Mota Filho et al. 2011).

Phase 2: Monitoring of the oestrous cycle

Females were observed daily until a haemorrhagic vulval discharge was noted, and this was considered to be the first day of proestrus. From this moment, vaginal swabs were collected every 2 days for vaginal smears and analysis of the percentage of keratinized vaginal epithelial cells via light microscopy (Concannon and DiGregorio 1986; ×100–400; Opticus Co. Ltd., Olympus, Tokyo, Japan). The bitches were subjected to NB (Group 1) or AI with frozen/thawed semen (Group 2) from the same male when they had, respectively, ≥70% and 80% keratinization of the vaginal epithelium. The procedures for NB or AI were repeated once 36–48 h after the first mating (Group 1) or 24 h after the first AI (Group 2).

Hormone level assessment

In Group 2, blood samples (5 ml) were obtained at the time of the first AI by jugular venepuncture into heparinized tubes. These samples were centrifuged (1000 × g; 15 min) and the plasma stored at −20°C until the progesterone assay could be performed. The plasma progesterone concentration (ng/ml) was measured by radioimmunoassay.

Artificial insemination

Semen samples were thawed and evaluated as previously described. The aliquot used for insemination (2 ml) was re-diluted in 2 ml of ACP-106c (inseminating volume: 4 ml) and re-evaluated with respect to the subjective parameters of total motility and vigour; a total of 400 × 106 sperm cells were used. The females were inseminated using an Osiris® (IMV Technologies, L'Aigle, France) catheter, and once the catheter was removed, they were kept with their hindquarters elevated at 45–60° for 7 min.

Pregnancy diagnosis

Pregnancy diagnosis was performed between 25 and 35 days after the second mating or AI, using a portable ultrasound equipped with a micro-convex 5-MHz probe (ECHOVET 1500 Honda®; ECHO, Toyohashi, Aichi, Japan). The pregnancy rate (the number of pregnant females over the number of mated or inseminated females ×100) was subsequently calculated. The parturition rate (the number of litters over the number of pregnant females ×100) was calculated at birth, and the number of puppies was counted and litter sex ratio recorded.

Statistical analysis

Results were expressed as means ± standard error of mean. All of the percentage data were subjected to angular transformation prior to statistical analysis. Analysis of variance (anova), followed by Fisher's PLSD test, was used to determine the effect of semen processing (fresh or frozen/thawed semen) on motility, vigour, number of sperm cells with functional membrane, normal sperm cells, sperm cells with normal acrosomes, live sperm cells and litter size. The pregnancy and parturition rates were analysed by Fisher's exact test, and the proportion of males and females born from NB or AI was analysed using chi-squared tests. The comparison between the proportion of males and females born within the same group was analysed by anova followed by Fisher's PLSD test (Stat View for Windows®, Version 5.0, 1998, and sas; SAS Institute Inc., Cary, NC, USA). Values of p < 0.05 were considered to be significant.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The results obtained after thawing for the subjective analysis show that all parameters were lower (p < 0.05) than those observed for fresh semen, except sperm cells with normal acrosomes (Table 1). The results of the computer-assisted semen analysis (CASA; Table 2) showed excellent values for total motility (77.3%).

Table 1. Means (±SEM) of the subjective analyses of fresh and thawed semen (n = 10)
ParameterSemen
FreshThawed
  1. Different letters on the same row indicate significant differences (p < 0.05).

Volume (ml)1.0 ± 0.1
Concentration (108 sperm cells/ml)10.5 ± 1.1
Total motility (%)97.0 ± 1.5a77.0 ± 2.6b
Vigour (0–5)4.8 ± 0.1a3.8 ± 0.1b
Membrane functionality (%)87.2 ± 1.3a49.5 ± 1.3b
Normal sperm cells (%)85.6 ± 1.1a80.0 ± 1.9b
Sperm cells with normal acrosomes (%)92.9 ± 0.9a88.5 ± 2.0a
Live sperm cells (%)97.4 ± 0.3a47.1 ± 0.4b
Table 2. Means (±SEM) and the minimum and maximum values determined by Computer-Assisted Semen Analysis in thawed semen (n = 10)
ParametersValuesMinimum–Maximum
Total motility (%)77.3 ± 4.160.4–93.0
Progressive motility (%)29.7 ± 2.415.9–39.9
VCL (μm/s)32.6 ± 3.812.8–51.7
VSL (μm/s)22.6 ± 2.48.1–33.2
VAP (μm/s)20.4 ± 3.09.7–39.0
LIN (%)60.1 ± 1.352.4–67.2
STR (%)74.2 ± 1.964.4–83.3
ALH (μm)1.8 ± 0.21.0–3.1
BCF (Hz)5.4 ± 0.92.5–10.4
Rapid sperm cells (%)25.5 ± 4.46.1–45.4
Medium sperm cells (%)19.2 ± 1.86.6–26.3
Slow sperm cells (%)39.2 ± 3.527.6–64.9
Static sperm cells (%)16.1 ± 3.40.7–36.3

The percentage of keratinization of the vaginal epithelium on the first day of oestrus was 84 ± 2.2% for NB and 84.5 ± 2.8% for AI, which corresponded to 9.3 ± 0.3 and 11.8 ± 0.3 days from the onset of bleeding for groups NB and AI, respectively. The plasma progesterone concentration on the first day of AI was 7.9 ± 0.4 ng/ml.

Pregnancy and parturition rates for NB group were 100% with a mean litter size of 5.4 ± 0.3. Following AI, a 60% pregnancy rate was observed, resulting in a 50% parturition rate and a mean litter size of 3.4 ± 0.6 (Table 3).

Table 3. Reproductive parameters after natural breeding (NB, n = 10) and artificial insemination with thawed semen frozen in ACP-106c (ACP-106c, n = 10)
ProcedurePregnancy rate (%)Parturition rate (%)Litter sizeSex ratios of puppies (%)
MaleFemale
  1. Different superscript lowercase letters on the same column indicate significant difference (p < 0.05); uppercase letter on the same row (p > 0.05).

NB100 (10/10)a100 (10/10)a5.4 ± 0.3a2.6 ± 0.2 (48.2)Aa2.8 ± 0.3 (51.8)Aa
ACP-106c60 (6/10)b50 (5/10)b3.4 ± 0.6b1.6 ± 0.3 (47.1)Ab1.8 ± 0.4 (52.9)Ab

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The post-thawing results obtained were higher than those described by Rota et al. (2009) for the motility of semen frozen in Tris–egg yolk, using glycerol (62%) or ethylene glycol (51.6%) and by Nizański (2006) using Tris–yolk–glycerol with 1% Equex® (70.9% and for total motility).

The better results obtained with the current study compared with a previous study using the same extender (Cardoso et al. 2007) are perhaps because of the reduction in egg yolk concentration from 20% to 10%, thus favouring smaller amounts of lumps and better dissolution, with a consequently improved visualization of sperm cells. The choice to use 10% egg yolk in the present study was based on additional results obtained by our research team (Barbosa et al. 2009) using ACP-106c, which showed that this lower concentration is more practical for the analysis of sperm motility (objective or computerized) because of the lower viscosity of the medium, which facilitates sperm cell displacement. The differences between subjective and computerized analyses are associated with each observer's evaluation, although they also appear to depend on the dissolution of the egg yolk in the extender, given that the subjective and objective values of motility were similar when using Tris with 20% egg yolk and 6% glycerol (Silva et al. 2006b). The values obtained using CASA are directly correlated with those obtained by subjective analysis under light microscopy (Rijsselaere et al. 2005). The improved dissolution also promotes an improved protection against damage to the sperm membrane caused by the low temperature (Bouchard et al. 1990).

The parturition rate obtained in the present study differed from the pregnancy rate because of the occurrence of a miscarriage in one of the bitches, resulting from haematological changes caused by Ehrlichia canis. Reduced litter sizes when using intravaginal AI have already been observed with frozen/thawed semen (Nizański 2006), as was observed with the use of ACP-106c.

Although our team has shown that semen diluted in ACP-106c and cooled prior to insemination led to increased birth of female pups in the French Bulldog (Uchoa et al. 2012), the same effect was not observed in the present study, probably due to the too small number of pups to show any effect of gender.

Despite oestrus can already be considered to have begun when above 50% of anucleate cornified vaginal cells are observed, AI with frozen/thawed semen is currently considered to be reliable with over 80% keratinization.

The literature suggests that insemination should be performed approximately 3–5 days after the LH surge for AI using frozen/thawed semen, which quickly loses its viability (Silva et al. 1996). Values between 12 and 19 ng/ml of progesterone have been reported as ideal for intravaginal AI with frozen/thawed semen (Rota et al. 2009). However, there are no laboratories in our region capable of performing progesterone assays and providing results in <2 days, nor were we able to use progesterone assay kits because of their cost, thus making it impossible to use progesterone-based AI. The pregnancy rate of AI with frozen/thawed semen is lower than that obtained with NB, regardless of the site of deposition, because frozen/thawed semen has lower viability (Fontbonne and Badinand 1993) and often fails to fertilize ovulated oocytes (Rota et al. 2009). Even though the good results obtained for the pregnancy rate from intravaginal AI in the present study (60%) are similar to those observed by Fontbonne and Badinand (1993) with Tris: 52.6%; Silva et al. (1996) with laiciphos: 60%; and Nizański (2006) with Tris: 60.7% and notwithstanding the excellent results described by Rota et al. (2009) with Tris–yolk–glycerol (100%) and ethylene glycol (100%), we believe that the results for the pregnancy rate obtained in the present study, with the use of ACP-106c–yolk–glycerol, can be further improved. Importantly, the present study is the first one using AI with frozen/thawed canine semen and powdered coconut water (ACP-106c). This report opens perspectives for further studies evaluating the use of ACP-106c with the determination of the ideal moment for AI through progesterone analysis, and with other cryoprotectants, different freezing and thawing curves, or other sites for semen deposition. The results of intrauterine AI, either through laparoscopy, with semen deposition directly in the uterine horn, or through a trans-cervical Norwegian catheter, are 75% (Thomassen et al. 2006) to 100% (Silva et al. 1996). The decision to use intravaginal AI in the present study was because of the practicality of the method and the feasibility of future implementation of the commercial use of ACP-106c, given the ethical aspects and difficulties involved with the implementation of routine intrauterine AI that involves sedation/anaesthesia and/or surgery.

This study shows that ACP-106c can be successfully used to freeze dog semen and that the vaginal deposition of such semen may yield pregnancy rates similar to those obtained with other studies.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

We thank ACP Biotecnologia for providing the ACP-106c extender; ‘Grande Canafístula’ kennel for allowing their animals to be used in the study; Royal Canin for providing the HT-42 dog food used; and CAPES, FUNCAP and CNPq for their financial support.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Lúcia Daniel Machado da Silva designed the experiment. Daniel Couto Uchoa and Antônio Cavalcante Mota Filho were responsible for semen collection, semen processing, semen evaluation, monitoring pregnancy and birth, and artificial inseminations. Ticiana Franco Pereira da Silva analysed the data. Daniel Couto Uchoa, Lúcia Daniel Machado da Silva and Ticiana Franco Pereira da Silva drafted the paper. Lúcia Daniel Machado da Silva revised critically the manuscript.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References
  • Barbosa CC, Madeira VLM, Jucá RP, Oliveira AC, Uchoa DC, Silva LDM, 2009: Cryopreservation of canine semen using a powdered coconut water extender (ACP-106®): effect of glycerol temperature addition (27 and 4°C). Ciência Animal Brasileira 10, 12091217.
  • Bouchard GF, Morris JK, Sikes JD, Youngquist RS, 1990: Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoa motility. Theriogenology 34, 147157.
  • Cardoso RCS, Silva AR, Silva LDM, Chirinéa VH, Souza FF, Lopes MD, 2007: Evaluation of fertilizing potential of frozen-thawed dog spermatozoa diluted in ACP-106 using an in vitro sperm-oocyte interaction assay. Reprod Dom Anim 42, 1116.
  • Concannon PW, DiGregorio GB, 1986: Canine vaginal cytology. In: Burke TJ (ed.), Small Animal Reproduction and Infertility. Lea & Febiger, Philadelphia, pp. 96111.
  • Fontbonne A, Badinand F, 1993: Canine artificial insemination with frozen semen: comparison of intravaginal and intrauterine deposition of semen. J Reprod Fertil Suppl 47, 325327.
  • Mota Filho AC, Teles CHA, Jucá RP, Cardoso JFS, Uchoa DC, Campello CC, Silva AR, Silva LDM, 2011: Dimethylformamide as a cryoprotectant for canine semen diluted and frozen in ACP-106C. Theriogenology, 76, 13671372.
  • Nizański W, 2006: Intravaginal insemination of bitches with fresh and frozen- thawed semen with addition of prostatic fluid: use of an infusion pipette and the Osiris catheter. Theriogenology 66, 470483.
  • Rijsselaere T, Van-Soon A, Tanghe S, Coryn M, Maes D, Kruif A, 2005: New techniques for the assessment of canine semen quality: a review. Theriogenology 64, 706719.
  • Rota A, Milani C, Romagnoli S, Zucchini P, Mollo A, 2009: Pregnancy and conception rate after two intravaginal insemination with dog semen frozen either with 5% glycerol or 5% ethylene glycol. Anim Reprod Sci 118, 9497.
  • Silva AR, Cardoso RCS, Silva LDM, 2006a: Influence of Temperature during Glycerol Addition and Post-thaw Dilution on the Quality of Canine Frozen Semen. Reprod Dom Anim 41, 7478.
  • Silva AR, Cardoso RCS, Silva LDM, Chirinéa VH, Souza FF, 2006b: Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions. Theriogenology 66, 456462.
  • Silva LDM, Onclin K, Lejeune B, Verstegen JP, 1996: Comparisons of intravaginal and intrauterine insemination of bitches with fresh or frozen semen. Vet Rec 138, 154157.
  • Thomassen R, Sanson G, Krogenaes A, Fougner JA, Berg KA, Farstad W, 2006: Artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach. Theriogenology 66, 16451650.
  • Uchoa DC, Silva TFP, Cardoso JFS, Mota Filho AC, Jucá RP, Silva AR, Silva LDM, 2012: Favoring the birth of female puppies after artificial insemination using chilled semen diluted with powdered coconut water (ACP-106c). Theriogenology 77, 19591963.