MATERIALS AND METHODS

Study area: Study was conducted at University Livestock Farm of CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur (32.6°N, 76.3°E, altitude 1290.8 m) from September-December, 2017, January and March, 2018 and September-December, 2018, respectively.

Research procedure: The study was conducted on apparently healthy Gaddi bucks (n = 11, aged 2.16±0.36 years, weighing 39.1±2.82 kg), Chegu bucks (n = 8, aged 1.98±0.34 years, weighing 28.1±1.96 kg). These bucks were selected on basis of breeding history, breeding soundness evaluation and testicular diameters. All the bucks were maintained under identical conditions and were screened for diseases, Brucellosis⁸, Chlamydiosis⁹ to eliminate the possible transmission of infection.

Average light: dark h (h) and temperature (°C) during the period of study comprised the average of the values considered on the day of semen collection and the preceding days. The average light:dark h (h) and temperature (°C) ranged from 7.07±0.41:16.93±0.41 to 12.29±0.13: 11.71±0.13 and 9.88±0.51 to 21.94±0.18°C, respectively. Similar group of bucks from both the breeds were utilized for semen analysis during the period under study.

All bucks were subjected to grazing for 5 h and remained under confinement in a shed where they were fed as per the standards of Indian Council of Agricultural Research (ICAR)¹⁰. All males had round the clock access to the clean drinking water.

A total of 286 ejaculates (Gaddi, n = 106, Chegu, n = 180) from adult healthy bucks were collected using an estrus doe. The collections were made twice weekly in artificial vagina (AV) maintained at 42-43°C. The fresh ejaculates were evaluated for any gross abnormality including color as well as volume and concentration (Caprine Photometer, IMV 1409^®) followed by microscopic examination for mass motility. The suitability of ejaculates for further processing was done using the standard criterion¹¹. Absence of any gross abnormality, milky to creamy semen color, mass motility of >3 and initial progressive motility of >70% were the criteria for selection of semen. The post- thaw cryopreserved semen samples were evaluated for progressive motility¹², viablility¹³, morphological abnormalities¹⁴ and HOST reactivity¹⁵. Percent change in the aforesaid parameters between freshand post-thaw semen was also calculated to determine the effect of cryopreservation.

The seminal plasma was removed as described previously^16,17. The semen pellet thus obtained was extended with 2 equal fractions of tris citrate egg yolk extender, TEY (TRIS 1.21 g, Citric acid 0.685 g, D-fructose 0.5 g, benzyl penicillin 1000 IU mL^–1, streptomycin sulphate 1 mg mL^–1), EY (10%) and glycerol (6%). The TEY was added to semen at a gap of 2-3 min to yield a final concentration 150×10⁶ spermatozoa/straw. The pH of the buffer was adjusted to 6.7-6.9.

Extended semen was filled in 0.25 mL French mini straws (IMV Technologies, L’Aigle, Cedex, France), by aspiration using micropipette (Minitube, Germany) and subsequently sealed at free end with the help of polyvinyl alcohol (PVA) (IMV Technologies, L’Aigle, Cedex, France) powder. All the aforementioned steps of semen handling were undertaken at controlled air temperature of 30°C. The straws were laid on a stainless steel rack and placed in cooling cabinet 4°C (Macro Scientific works Pvt. Ltd. India) for 4 h and thereafter exposed to liquid nitrogen vapors for 7 min. Finally the straws were plunged into liquid nitrogen for storage. The inventory of semen storage was also maintained. Thawing of semen straws was done at 37°C for 30 sec in a water bath¹⁸.

Proper ethical considerations related to animal handling and semen collections were observed and ensuring not to cause any injury during sampling.

Statistical analysis: The data obtained were analyzed using package¹⁹ R version 3.4.3. Multivariate analysis was used to determine correlations between climatic condition (Temperature (°C) and Sunshine (h) with the fresh and post-thaw semen quality parameters and to frame regression equations and their significance was tested again by using ANOVA. Results were presented as Mean±SEM and differences were considered significant when p<0.05.

RESULTS AND DISCUSSION

Correlations between the climatic conditions (Temperature (°C), sunshine (h)) and seminal parameters (progressive motility, live and dead, morphological abnormalities and HOST reactivity) are presented in Table 1. Amongst the progressive motility, viability, morphological abnormalities and HOST reactivity, it was only the HOST reactivity that was negatively and significantly correlated (r = -0.471, r = -0.527, p<0.01) to temperature in both fresh diluted and post thaw semen in Gaddi goats (Table 1). It can be inferred that with reduction in temperature there is improvement in HOST reactive sperms in Gaddi buck semen. Whereas, temperature was negatively significantly correlated (r = -0.745, r = -0.768, p<0.01), with post thaw progressive motility and viability in Chegu goats (Table 1) which suggests better post thaw semen quality during the winter months.

Present study evaluated relationship of fresh diluted and post thaw semen parameters to change in climatic conditions within the short days of breeding season, which however is not available in the literature. Irrespective of the lack of literature for direct comparison, the present study delineates temperature rather than sunshine (h) affects semen quality. During low temperature of breeding season there was an improvement in HOST reactivity in fresh and post thaw semen in Gaddi bucks. Similarly, during a period of low temperature the post thaw progressive motility and viability were higher in Chegu buck semen. Murase and coworkers reported that post thaw motility was significantly higher in winters while HOST reactivity did not differ between season²⁰. Moreover the relation to temperature to post thaw betterment of motility and viability in Chegu bucks could also be due to the reason that Chegu goat is native of temperate reason and may therefore perform better under low temperature.

Spring and summer have been generally accepted by various researchers’ to reduce the seminal quality with increased percentage of dead and abnormal sperms along with reduced motility^21-22. Extreme temperature affects the function of the epididymis, alters the semen pH towards alkalinity²³ and increases the percentages of abnormal and dead sperms²⁴ and could also lead to testicular degeneration and even complete sterility^23,24.

On the other hand, the aforesaid trend on semen quality revealed, autumn to be the best season resulting in better motility, viability and morphology of sperms, along with enhanced ability to withstand cryo-injuries^25,26. Kumar and coworkers observed better ability of sperms to withstand freezing and thawing during autumn than winter²⁷.

Winters have been equivocal in affecting the semen quality. Researchers observed better semen quality during winters⁵. Similarly, Gallego-Calvo⁶ and coworkers reported increased post thaw quality but reduced percentage of motile sperms during winter. Other studies corroborated winter to favour semen quality^28,29. Reduced percent of dead sperms were observed during winters^24,26. Researchers observed no effect on seasonal variability on HOST reactivity, while the post thaw motility, acrosomal integrity and abnormalities were severely affected in autumn compared to winter²⁰. To the contrary, the desired semen quality could not be achieved in winters^30,31.

Delgadillo and coworkers suggested photoperiod as main factor responsible for seasonality in buck reproductive activity. Short days and decreasing day length stimulate the secretion of luteinizing hormone (LH), which intern, induces testicular growth and the release of testosterone, resulting in quantitative and qualitative improvements in semen production along with increased sexual behavior³². Contrastingly, long days and increasing day length reduce LH secretions and testicular growth, leading to a fall in the plasma testosterone concentrations, reduced sperm quality and diminished sexual behavior^21,22.

However other workers also propose that it is not only photoperiod that solely regulates but several other factors which may also influence semen. Hence there is a consensus among researchers interested in breeding of goats, that the quality of bucks’ semen is affected by various factors including but not limited to breed³, season^{6,26,30,32,33} and source of protein supplemented in ration³⁴. Whereas, another group of researchers argued that season effects on semen quality were minor and the semen quality remained in the range of good semen in all seasons³⁵. Some studies have shown that the sperm motility of pubertal bucks was high in autumn and low in summer season²⁴.

In addition to effect of photoperiods on the LH release as listed wide supra, a favorable season for semen production could be attributed to changes in total protein and protein composition of seminal plasma³⁶. Seminal plasma protein may influence seasonal resistance of the spermatozoa to freeze thaw damage. Smith and coworkers recorded higher total protein concentration of seminal plasma (36.9 vs. 23.0 mg mL^–1) during breeding than non-breeding season suggesting its role in protection against cryopreservation effects³⁶. Researchers observed that sperm samples supplemented with proteins from the buck seminal plasma obtained during non-breeding season led to deterioration of sperm quality³⁷, while other studies have demonstrated the positive effects on sperm quality after thawing when the semen was supplemented with proteins or seminal plasma collected during breeding season³⁸. There were alterations in total protein composition of seminal plasma that influenced seasonal resistance of semen to freeze thaw process.

CONCLUSION

It was concluded that temperature rather than sunshine (h) is the main factor affecting the seminal quality parameters in both Gaddi and Chegu breed of goat during breeding season.

SIGNIFICANCE STATEMENT

This study discovered the correlation of climatic parameters with seminal quality in Gaddi and Chegu goats of Himachal Pradesh, India. In Gaddi goats, HOST reactivity was negatively and significantly correlated to temperature in both fresh diluted and post thaw semen. Whereas, temperature was negatively significantly correlated with post thaw progressive motility and viability in Chegu goats. In brief, study suggests temperature as the main factor affecting the seminal quality parameters in both Gaddi and Chegu breed of goat during breeding season. The study will help the researchers to uncover the relationship of climatic factors with seminal quality in different breeds reared in different geographical locations and strengthen our finding of impact of temperature rather than sunshine (h) on the seminal parameters in goats.

ACKNOWLEDGMENTS

Authors are grateful to Dr Madhumeet Singh (Professor and Head, Department of Vety. Gynae.), Dr. Mandeep Sharma (Dean, College of Veterinary and Animal Sciences) CSKHPHV, Palampur for technical and administrative support and Department of Dairy and Fisheries, GOI for financial assistance under project Adhoc-Misc 2127-37 for carrying out this study.