Effect of Natural Photoperiod on Epididymal Sperm Quality and Testosterone Serum Concentration in Domestic Cat (Felis silvestris catus)

Authors


Author's address (for correspondence): María Alejandra Stornelli, Laboratorio de Reproducción Animal, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Calle 60 y 118, La Plata B1900AVW, Buenos Aires, Argentina. E-mail: astornel@fcv.unlp.edu.ar

Contents

The aim of this study was to assess epididymal sperm characteristics and serum testosterone concentration in cats under natural photoperiod. The hypothesis was that natural photoperiod induces seasonal changes in spermatozoal quality and serum testosterone concentration. Mixed breed tomcats (n = 43) that underwent bilateral orchiectomy at a municipal public pet shelter were used in the study. Epididymides were divided into two groups according to time of castration. In Group I, toms were castrated during increasing light (IL; [winter and spring; n = 24]), and group II, during decreasing light (DL; [summer and fall; n = 19]). Only mature toms castrated in the two lasts weeks of each season were included in this study. Sperm samples were obtained by cutting the cauda epididymis in Tris solution and tested for motility (MOT,% motile), velocity (VEL, 0–5), total sperm cells (TS, 106), acrosome integrity (ACR,% intact; FITC-PSA), plasma membrane integrity (MI,%intact; CFDA-PI) and sperm morphology (SM,% normal). Before orchiectomy, blood samples were taken to measure serum concentrations of testosterone (T2) by a solid-phase RIA. Data were analysed with the mixed procedure of SAS. Toms castrated during IL had higher sperm plasma membrane integrity and better sperm morphology compared to toms castrated during DL (69.0 ± 2.7 vs 60.6 ± 2.1, p < 0.01; 45.9 ± 2.5 vs 35.9 ± 3.4; p < 0.02; respectively) and tended to have higher sperm motility and total number of sperm cells compared to toms castrated during DL (56.3 ± 2.8 vs 47.3 ± 3.7, p < 0.06; 13.8 ± 1.4 vs 10.0 ± 1.8, p < 0.09). However, velocity, acrosome integrity and serum testosterone concentrations were similar between both groups (3.5 ± 0.1 vs 3.4 ± 0.1, p > 0.6; 45.8 ± 3.3 vs 44.0 ± 4.0, p > 0.72; 0.76 ± 0.15 vs 0.59 ± 0.19, p > 0.51; respectively). In conclusion, natural photoperiod induces seasonal changes in sperm quality with a moderate variation in serum testosterone concentrations.

Introduction

The queen is a seasonal breeder when exposed to natural photoperiod, with ovarian activity ceasing under decreasing photoperiod and resuming with increasing photoperiod. This seasonality may be observed in both females and males of several mammalian species, and in these species, photoperiod and melatonin concentrations are related to circannual sperm production (Chemineau et al. 2008). This seasonality is observed in geographic locations where large differences between hours of light during the year are present and occurs as an adaptation to annual changes in the habitat (Malpaux et al. 2001; Chemineau et al. 2008). Although the reproductive features of queens are well known, in tom cats, there are few studies about sperm production. Likewise, the effect of season on reproductive function in the male domestic cat has been insufficiently explored. Whereas previous studies concluded that male cats are not seasonal in sperm production (Spindler and Wildt 1999; Franca and Godinho 2003), more recent studies challenge those findings and suggest seasonality of sperm production (Axner and Linde Forsberg 2007; Blottner and Jewgenow 2007; Stornelli et al. 2009). Previous studies have shown seasonal changes in testicular cell morphology in domestic cats (Stornelli et al. 2009). Probably, these changes are reflected in seasonal variations of epididymal sperm quality. In the same way, seasonal mammals show changes in testosterone serum concentrations during the year related to sperm production and cycles of testicular growth and involution (Johnstone et al. 1984; Blottner and Jewgenow 2007). The aim of this study was to assess epididymal sperm characteristics and serum testosterone concentration in cats under natural photoperiod. Our hypothesis was that natural photoperiod induces seasonal changes in spermatozoa quality and serum testosterone concentration.

Materials and Methods

Forty-three privately owned short-hair mixed breed male cats aged between 1 and 5 years were included in this study. The cats were included in a programme for breeding control at the Municipal Public Pet Shelter of the City of La Plata. Before surgery, all animals were anesthetized with a combination of ketamine (25 mg/kg im; Vetanarcol®, Laboratorios Koning SA, Avellaneda, Buenos Aires, Argentina), xylazine (1 mg/kg im; Sedomin®, Laboratorios Koning SA, Avellaneda, Buenos Aires, Argentina). After surgery, all animals were medicated for 3 days with amoxicillin (22 mg/kg im, Clamoxyl LA, Laboratorio Pfizer SRL, Ciudad Autónoma de Buenos Aires, Argentina) and tramadol (1 mg/kg im; Algen® Laboratorio Richmond SA, Grand Bourg, Buenos Aires, Argentina). All surgical procedures were performed by a licensed veterinarian and followed approved guidelines for ethical treatment of animals (CIOMS 1985). To show the true effect of season, only toms castrated during the two lasts weeks of each season were included in the study (winter: second and third weeks of September [09/06/10 to 09/20/10]; spring: second and third weeks of December [12/06/10 to 12/20/10]; summer: second and third weeks of march [03/06/10 to 03/20/10]; fall: second and third weeks of June [06/06/10 to 06/20/10]). Hence, according the time of castration, epididymides were divided into two groups. Group I toms were castrated during increasing light (IL; [winter and spring; IL, n = 24]), and group II toms were castrated during decreasing light (DL; [summer and fall; IL, n = 24]). After orchiectomy, each testis with adjacent epididymis was transported to the laboratory in saline solution. Right and left epididymides were processed separately, and epididymal sperm recovery was made within 1–2 h after surgery. For recovering epididymal sperm, the cauda epididymis was first carefully separated from the testis and ductus deferens and placed in a glass tube containing 0.75 ml Tris solution (3.02 g Tris, 1.70 g citric acid, 1.25 g fructose, distilled water added up to 100 ml). After 10 min at 37°C, sperm samples were obtained by cutting the cauda epididymis in Tris solution. The following tests were performed on sperm samples: motility (MOT,% motile), velocity (VEL, 0–5), total sperm cells (TS, 106), acrosome integrity (ACR,% intact; FITC-PSA), plasma membrane integrity (MI,%intact; CFDA-PI) and sperm morphology (SM,% normal; Tincion 15®, Biopur, Rosario, Santa Fe, Argentina). Before bilateral orchiectomy, blood samples were taken to measure serum concentrations of testosterone (T2). All samples were centrifuged and stored at −20°C until T2 were measured by a solid-phase radioimmunoassay (RIA) using I125 (Coat-A-Count, Total Testosterone; Diagnostic Product Corporation, Los Angeles, CA, USA). Data were analysed with the mixed procedure of SAS. Hence, sperm quality and testosterone concentrations of toms castrated in days with increasing light (IL, 9 h 51’ to 14 h 27’ daylight; Group I) were compared with toms castrated in days with decreasing light (DL, 14 h 27’ to 9 h 51’ daylight; Group II).Data are presented as LSM ± SEM. Significance was defined as p < 0.05.

Results

Toms castrated during IL had higher sperm plasma membrane integrity and sperm morphology compared to toms castrated during DL (69.0 ± 2.7 vs 60.7 ± 2.1, p < 0.01; 45.9 ± 2.5 vs 35.9 ± 3.4; p < 0.02; respectively). Similarly, toms castrated during IL tended to have higher sperm motility and total sperm counts compared to toms castrated during DL (56.3 ± 2.8 vs 47.3 ± 3.7, p < 0.06; 13.8 ± 1.4 vs 10.0 ± 1.8, p < 0.09; respectively). However, velocity and acrosome integrity were similar between the two groups (3.5 ± 0.1 vs 3.4 ± 0.1, p > 0.69; 45.8 ± 3.3 vs 44.0 ± 4.0, p > 0.72; respectively). Serum testosterone concentrations were similar between toms castrated in IL vs toms castrated in DL (0.76 ± 0.15 vs 0.59 ± 0.19, p > 0.51).

Discussion

Some studies concluded that male cats are not seasonal in sperm production (Spindler and Wildt 1999; Franca and Godinho 2003). However, it is well known that the queen is a seasonal breeder when exposed to natural photoperiod, with ovarian activity ceasing under decreasing photoperiod and resuming with increasing photoperiod. Melatonin secretion is controlled by the prevailing photoperiod, with higher concentrations during the dark phase. This seasonality may be observed in both females and males of other mammal species (Chemineau et al. 2008). In those, photoperiod and melatonin concentrations are related to circannual sperm production. Recent studies show seasonal changes in testicular cell morphology in domestic cat (Stornelli et al. 2009). Stornelli et al. (2009) found that testicles from males orchiectomized during long hours days had a higher percentage of tubules with tailed sperm and mature sperm (ready to be release from Sertoli cells) compared to testicles from males orchiectomized during short hours days. In agreement, testicles from males orchiectomized during long hours days had a lower percentage of round and elongated sperm compared to testicles from males orchiectomized during short hours days (Stornelli et al. 2009). It has been reported that the number of sperm collected by electroejaculation was different between the non-breeding seasons and breeding seasons of male cats in Australia (Johnstone 1984). Similarly, Blottner and Jewgenow (Blottner and Jewgenow 2007) have found that testis weight and the total sperm number per testis showed significant differences between spring and autumn. These findings support our result in which we found a tendency to have higher motility and total sperm cells in increasing light compared with decreasing light.

Axner and Linde Forsberg (2007) found a higher percentage of normal spermatozoa during February to July than during August to January. This result is in agreement with our findings that show that toms castrated during IL had higher plasma membrane integrity and sperm morphology compared to toms castrated during DL.

The range of testosterone blood concentration found in toms castrated in IL in this work was similar to those reported by Tsutsui et al. (2009) during the breeding season (0.25–2.5 ng/ml, with an average of 1.69 ng/ml). Whereas Johnstone et al. (1984) reported that testosterone levels were greatly affected by the season, Kirkpatrick (1985) reported hardly any differences between breeding and non-breeding season in agreement with the results from this study.

Conclusion

Natural photoperiod induces seasonal changes in spermatozoa quality but not in serum testosterone concentrations.

Acknowledgements

We wish to thank the Municipal Public Pet Shelter for cooperating with our research programme. Also, we thank the Veterinary Immunoparasitology Laboratory for the use of their fluorescence microscope. This study was supported in part by UNLP grant V11/162 and V11/200 to RLS and MAS. In addition, R Nuñez-Favre was supported with a scholarship from CONICET, MC Bonaura with a scholarship from CIC and D Mansilla-Hermann with a scholarship from UNLP.

Conflicts of interest

None of the authors have any conflicts of interest to declare.

Author contributions

Nuñez-Favre R helped to design, conduct the experiment and process the samples, analysed data and drafted manuscript. Bonaura MC, Tittarelli CM and Mansilla-Hermann D helped to conduct the experiment and process the samples. De la Sota RL and Stornelli MA helped to design and conduct the experiment and critically revised the manuscript.