Seasonal Influence on Post Thaw Seminal Attributes and Conception Rate from Semen of Jaffarabadi Buffalo Bulls

Artificial insemination (AI) with frozen thawed semen in buffalo has been reported to have low success rate than in cattle because of poor freezability and fertility (Kumaresan et al., 2005). So that the quality of the frozen thawed semen is one of the most powerful factors affecting the conception (Saacke, 1984). The production and reproduction are impaired as a result of drastic changes in biological functions caused by heat stress (Marai et al., 1995). High ambient temperature during summer adversely affects testicular size, libido and semen quality (Soderquist et al., 1996). The existence of a biological mechanism which makes them sensitive to the breeding stimulus during a particular season of the year is well established, but the exact nature of the internal mechanism that controls seasonal responses is not known. Most of the tests that are used for evaluation of semen are based on physical characters of spermatozoa. However, very little information is available on effect of environmental variables like temperature, humidity and day length in a particular season (Tiwari et al., 2011). Season may have its effect on seminal attributes, freezability and fertility. Therefore, present study was undertaken to investigate the effect of winter and summer seasons on post thaw seminal attributes and conception rate from semen of Jaffarabadi buffalo bulls.

The present study was carried out on semen ejaculates (192) collected by artificial vagina method in morning hours from four Jafarabadi breeding bulls and cryopreserved at Frozen Semen lab of Cattle Breeding Farm, JAU, Junagadh, Gujarat during November 2017 to October 2018. The animals were fed as per the minimum standard protocol (MSP-2000) decided by Government of India. The bulls were kept in sheltered paddocks with access to ad libitum water. The optimum health care was taken including clipping of preputial hair and vaccination against Haemorrhagic Septicaemia, Black Quarter and Foot and Mouth Disease. Besides this, screening for Tuberculosis, Johne’s disease, Brucellosis, Campylobacteriosis and Trichomoniasis was also carried out. The post thaw seminal traits were assessed after 24 hours of cryopreservation. A light microscope was used to determine post thaw activity (% score) and the percentage of individual spermatozoa depicting a pattern of progressive, rectilinear movement. Sperm concentration as million per ml of semen was estimated using bovine photometer with diluter and printer (Accucel, IMV, France) against 530 nM wave length. The morphological abnormalities of the spermatozoa were studied in the eosin-nigrosin stained slides. The acrosome integrity was evaluated by simplified nigrosine-eosin-Giemsa staining technique as described by Kutty et al., (1996), whereas the hypo-osmotic swelling test (HOST) was done to evaluate the functional integrity of the sperm membrane, based on curled and swollen tails as described by Jayendran et al., (1984). For freezing of semen Andromed extender and French medium straws were used employing conventional wide mouth liquid nitrogen freezer. The trial on first AI conception rate was carried out on 800 Jafarabadi buffaloes inseminated with frozen thawed semen of four bulls at Cattle Breeding Farm, JAU, Junagadh as well as in field during study period. The inseminated buffaloes were confirmed for pregnancy by per rectal palpation at 3 months post-insemination. The data was analyzed for calculating mean ± SE and significance by Statistical Packages for Social Sciences (IBM® SPSS® statistics, USA, version 20.0) software.

Post thaw motility
 
The mean per cent post thaw motility was 58.85±0.56 and 56.35±0.43 during winter and summer seasons, respectively. Statistical analysis revealed that the per cent post thaw motility differed highly significant (P<0.01) between the winter and summer season (Table 1). The per cent post thaw motility was found to be significantly higher (P<0.01) during the winter season as compared to summer season. Similar results in Murrah (Tiwari et al., 2011) and Surti bulls (Bhosrekar et al., 1992) have been reported, whereas Ram et al., (2019) did not find such significant difference in Murrah bulls.
 

       
Post thaw motility was very good in winter and poor in summer season because the most logical explanation is that spermatogenesis benefits from the cooler temperatures, or that the temperatures at processing are less difficult to maintain in winter than during the other seasons (Rasul et al., 2001). Summer spermatozoa may be fragile and cannot withstand freezing stress. No single attributes of semen can be relied upon for predicting fertility status of an ejaculate, however estimation of post thaw motility is increasingly being used as yardstick to assess the success of freezing technology and freezability of buffalo bull semen. Though a single sperm is able to fertilize the ovum but millions of sperm are required to counteract the uterine environment and to cross many hurdles to reach the site of fertilization. Thus giving more emphasis on post-thaw sperm recovery is an absolute necessity.
 
Post thaw live sperm count
 
The per cent post thaw live sperm count was observed to be 67.06±0.76 and 65.62±0.75 during winter and summer season, respectively. However, per cent post thaw live sperm which remained non-significantly higher during winter season compared to summer season (Table 1). The non-significantly higher per cent post thaw live sperm observed during winter season than summer season corroborated with earlier findings in Swamp buffalo bulls (Das et al., 2017) and Murrah bulls (Ram et al., 2019). While, significant difference was reported in Murrah bull (Nitharwal, 2013). This shows that the winter season protects sperm viability better during cryopreservation in comparison to other seasons. These results are consistent with previous studies in riverine buffaloes, which confirmed that spermatozoa survive freezing better during winter than summer (Mandal et al., 2000). Contrary to the present findings, Bhosrekar et al., 1992 reported the highest (79.80%) percentage of live sperm in summer and lowest (67.80%) in winter season; this might be due to different geographical location, different breed, experimental design and protective measure provided during experimental period.
 
Post thaw abnormal sperm count
 
The per cent post thaw abnormal sperm was 8.62±0.43 and 9.08±0.51 per cent, during winter and summer seasons, respectively. The post thaw abnormal sperm count was observed non-significant higher during summer compared to winter season (Table 1). The significant seasonal differences in post thaw abnormal sperm have been reported in Murrah bulls (Ram et al., 2019). The higher ambient temperature and humidity might have caused an adverse effect on spermatogenesis and negative effect on LH secretion (Gilad et al., 1993). During semen processing (dilution, equilibration and freezing) the abnormal sperm count increases (Nath et al., 1991). However, post-thaw abnormal sperm count remained within the permissible limit in the present study.
 
Post thaw HOS reactive spermatozoa
 
The mean per cent PHOST reactive spermatozoa were 58.52±0.67 and 55.43±0.60 during winter and summer seasons, respectively. Statistical analysis revealed that the per cent post-thaw HOST reactive spermatozoa were found to differ significantly (P<0.01) between the seasons (Table 1) and showed higher value in winter compared to summer season. Statistical analysis revealed that the per cent PHOST reactive spermatozoa were observed to be significantly higher during winter as compared to summer season. The present findings closely corroborated with the reports in Murrah bulls (Ram et al., 2019). However, Salah et al., (1992) reported non-significant effect of season on per cent post-thaw HOST reactive spermatozoa in HF bull. Assessment of post thaw sperm membrane function appears to be a significant marker for the fertilizing capacity of spermatozoa, since it is involved in metabolic changes with the surrounding medium and in the process of capacitation, acrosome reaction and fusion with the oocyte membrane.
 
Post thaw acrosome integrity of the spermatozoa
 
The per cent post thaw acrosome integrity of spermatozoa during winter and summer seasons were 75.22±0.27 and 75.29±0.22, respectively. Statistically, per cent post thaw acrosome integrity was non-significantly higher during winter as compared to that of the summer season (Table 1). Similar observations have been made recently in Murrah bulls (Ram et al., 2019). However, significant seasonal variation in the per cent post-thaw acrosomal integrity has been reported in crossbred bulls by others (Mishra et al., 2013; Bhakat et al., 2014). In the present study, slightly lower acrosome integrity observed summer than the winter season might be due to the damage during dilution, cooling, freezing and thawing processes.
 
First AI conception rate
 
The mean first AI conception rate was observed to be 45.75±0.21 (183/400) and 43.50±0.11 (174/400) during winter and summer season, respectively (Table 1). The first AI conception rate was significantly higher in winter compared with summer season (Table 1).
       
The non-significantly higher conception rate obtained in winter compared with summer in the present study very well corroborated with different reports in buffaloes (Das et al., 2017; Abdullah et al., 2017). The conception rates in artificially inseminated bovines varied considerably across species, breeds and geographical locations. These could have been due to multiple animals, environmental factors, expression and detection of heat symptoms, artificial insemination worker, semen handling from collection, processing to cryopreservation and thawing, individual dam effect and embryonic mortality.
       
The seasonal variations of conception rate might be due to differences in stress levels during different seasons as well as availably of grass. Heat stress directly affects fertility by reducing oocyte quality and indirectly by reducing feed intake thereby causing negative energy balance in animals and negatively affects reproductive performance by reducing quality of oocyte and embryo. Buffaloes have black body coat and less efficient evaporative cooling owing to poor sweating ability as they possess less number of sweat glands (Marai and Haeeb, 2010), which might contribute more heat load during summer season. Our findings are in line with Dash et al., (2015), who found depression in conception rate in Murrah buffaloes during the period of heat stress (summer).

The semen quality of post thaw samples were observed better during winter compare to summer season with satisfactory first AI conception rate in Jaffarabadi buffaloes.

The authors are thankful to the authorities of Cattle breeding Farm, JAU, Junagadh and Junagadh Agricultural University, Junagadh for granting permission to perform the research and providing necessary facilities.