Semen Production and Semen Quality of Mehsana Buffalo Breed Under Semiarid Climatic Conditions of Organized Semen Station in India

Semen production data comprising of 55071 ejaculates of 144 bulls from Mehsana buffalo breed was analysed. The traits under study were semen volume, sperm concentration, initial sperm motility, post-thaw sperm motility and number of semen doses per ejaculate. The objective of the present study was to assess the effect of various factors affecting semen production traits and measure the semen production potential of Mehsana buffalo bulls. Data collected of semen production traits were analysed using linear mixed model, including a random effect of bull along with xed effect of various non-genetic factors like farm, ejaculate number, season of birth, period of birth, season of semen collection and period of semen collection. First ejaculation had higher semen volume and sperm concentration resulted in to higher number of semen doses but semen quality was better in second ejaculation. Season of birth of the bull was affecting semen quality traits. As the period of birth advances semen volume increases whereas sperm concentration decreases which reected in persistent production of number of semen doses per ejaculate. Monsoon and summer were favorable seasons for semen collection because of higher sperm concentration which resulted in to higher semen doses per ejaculate. Additionally, Monsoon collected semen had highest volume. Hence, monsoon followed by summer season would be the favorable season for semen collection. Period of semen collection affecting all the semen production traits under study but it did not have specic trend which means managemental and environmental changes over the period have sizable inuence on the traits. Results of the study will help to plan future managemental practices and breeding strategies to improve semen production traits. Similar to the present Bhave in pooled data of Banni, Bhadawari, Pandharpuri and Surti buffaloes reported signicant effect of ejaculate on initial sperm motility.


Introduction
With an intensive selection for increased milk yield, reproductive performance was declined in many countries, in part due to an unfavourable genetic relationship. The intense selection for production traits in the last decades has led to a decrease in fertility.
Sustain or improve reproductive e ciency of dairy cattle along with productivity become one of the major challenges of the dairy industry worldwide. Many factors may account for decline in reproductive performance like physiological, nutritional, environmental and genetic. In this sense, several studies have recognized that there is substantial genetic variation underlying reproductive success in dairy cattle. More emphasis was given for improvement of reproduction traits in females only rather than the males. A de ciency in the breeding ability of the bull has a larger impact on the herd productivity as well as fertility problems of females The contribution of the males either through the natural mating or arti cial insemination (AI) cannot be ignored and careful scrutiny of the reproduction traits of bulls should be done before their extensive use in farm and eld condition for AI.
In recent years many countries have also implemented genetic evaluation for reproductive traits of the bulls. Thus, the relative emphasis of dairy cattle breeding has gradually shifted from production to functional traits such as reproduction. After extensive implementation of AI technique in eld condition, semen stations are working with the objective to maximize the output of good quality semen from bulls. To ful l the objective they are executing various semen evaluation method and also interested into factors affecting semen production traits of the bulls. This is assessed by aiming on semen volume, sperm concentration and sperm motility of the bull in each ejaculate. Moreover, scrutinizing the source of variations in semen production traits due to various non-genetic factors like farm, ejaculate number, season and period of birth, season and period of semen collection is necessary to ensure su cient semen production from the bulls.
Studies on semen production traits of buffaloes are very scanty (Singh et al. 2013;Bhakat et al. 2015; Ramajayan 2016: Bhave et al. 2020) as compared to cattle. Most of the studies on semen production traits of buffalo were done using very less number of ejaculates and focused on age of bull and primary managemental practices like semen collectors, time of collection and interval between collections. Location of the farm, season and period of birth, season and period of semen collection like non-genetic factors could have signi cant effect in variation of semen production traits. These non-genetic factors are associated with environment variation which might be came from feeding and other managemental practices at that time. The variation contributed due to these factors could be rectify after careful evaluation and semen production traits will be improved. The present study analyses semen production traits of two different semen stations of India with state of art facility for buffalo bull semen collection and processing.

Data
Evaluation of semen production traits was carried out on Mehsana buffalo bulls of two frozen semen stations of Gujarat viz. Pashu Samvardhan Kendra (PSK), Jagudan and Dama Semen Production Unit (DSPU), Dama. Information on breed characteristics is available on the national portal of the NBAGR website (National Bureau of Animal Genetic Resources 2021).
Data pertaining to semen production traits are available with two semen stations as database which were utilized for the present study with permission of the semen stations. Details of period of data, number of bulls and ejaculates utilized for the present study is given following table.

Semen collection and evaluation
Both the semen stations following standard routine practices for the collection of semen from Mehsana buffalo bulls. The bulls were cleaned properly on the day of semen collection in early morning before semen collection. For each bull semen collectors are speci ed and that semen collectors performed all the operations of semen collection for that particular bull. In the semen collection operations, Dummy bulls were used for sexual stimulus, and each bull allowed to perform 2 to 3 false mounts before nal semen collection ride. The time require for false mounting and actual collection mount varies from bull to bull. Normally one to three semen ejaculates were collected from the bull on the day of semen collection. After collection of semen, Semen volume was recorded and kept in a water bath at 37 °C. The semen stations are using photometer for estimation of sperm concentration per ejaculate (x 10 6 /ml). The sperm concentration was recorded per ml for particular ejaculate. The initial motility of the sperm cell was estimated by the semen stations as percentage by examining a drop of diluted semen with Tris buffer placed on a pre-warmed slide covered with a pre-warmed cover slip in a phase contrast microscope with a stage warmer at a magni cation of 40x. The sperm cells which exhibit progressive movement were scored on a scale of 0 to 100 percent. The collected semen with poor quality which did not ful l minimum standard criteria were removed from further process of frozen semen dose production. After completion of initial assessment, frozen semen doses were prepared using 0.25 ml straw which contain 20 × 10 6 sperms per dose (i.e., with the hypothesis that it reaches approximately 10 million motile sperms after thawing per dose), sealed, and printed. Semen straws were cooled at 4°C for approximately 3 hr after that frozen down at around −140°C for 10 min in a programmable freezer followed by storage in liquid nitrogen. Post-thaw sperm motility was then carried out for those stored frozen semen doses after 24 hr using 2-3 straws. Thawing of frozen semen straw was done by removing a straw from the liquid nitrogen container and plunging it in warm water bath at 37°C for 30 seconds. Frozen thawed semen was collected in a small test tube by cutting the ends of the straw and remaining procedure was as per initial sperm motility estimation.

Semen production traits and in uencing factors
Semen production traits considered to study the effects of various non-genetic factors are semen volume, sperm concentration, initial sperm motility, post-thaw sperm motility and number of semen doses per ejaculate.
Non-genetic factors affecting semen production traits are farm, number of ejaculate, season and period of birth, season and period of semen collection.
There are two farms under study. The bulls were maintained under proper housing, feeding, management and health care. The young bulls were trained for semen collection using arti cial vagina. The semen collection was done twice a week from individual bull and ejaculates were obtained with an interval of 15 -30 minutes. The nutrition requirement is standardized, so bulls are fed ad-libidum chaffed green and dry fodder mixture as per seasonal availability, concentrate mixture as per requirement based on body weight with area speci c mineral mixture. For analysis and description following coding is use.

Name of Farm Code
Pashu Samvardhan Kendra, Jagudan F1 Dama Semen Production Unit, Dama F2 Mehsana bulls were grouped as per seasons of birth of bull and season of semen collection as winter (November to February), summer (March to June) and monsoon (July to October) looking to the monthly average environmental conditions observed at farms. For analysis and description following coding is use. Semen collection from an individual bull was done two or three times in a day with the time interval of 15-30 minutes, accordingly it was grouped as rst (EJ1), second (EJ2) or third ejaculate. To study the effect of ejaculate number on various semen production traits in present study it was classi ed as such rst and second ejaculate as follow. Data pertaining to third ejaculate were limited, hence it was not utilized for the study.

Statistical Analysis
Abnormal records in semen production traits i.e. missing data or non-justi able data were eliminated. The non-genetic factors affecting the semen production traits of Mehsana buffalo bulls evaluated were farm, ejaculate number, season of birth, period of birth, season of semen collection and period of semen collection.
The effects of non-genetic factors on semen production traits like semen volume, sperm concentration, initial sperm motility, postthaw sperm motility and number of semen doses per ejaculate were studied by multivariate analysis under linear mixed model and restricted maximum likelihood (REML) method considering all non-genetic factors listed above have xed effect and bull as random effect to study the within bull and between bull variability. The data were analysed using SAS software version 9.3 and PROC MIXED as base command (2011).
The differences between the least squares means for sub classes under a particular effect were tested by Scheffe test (Scheffe, 1959) to check the signi cance. The high heterogeneous variances between the subclasses, unequal group size, pairwise and unpairwise comparison lead to use of Scheffe test as other tests nd differences between the least squares subclass means. Scheffe test is one of the best adjustments that can used to decrease experiment wise error rates when testing multiple comparisons. Scheffe test is a very conservative adjustment that why it is the safest method. The F-ratio used in the calculation is unique in that the mean square (MS) for only two groups being compared is used in the numerator and the MS for all respective comparison is used in the denominator. This means that each pairwise comparison has to have the same signi cance as the variance for all comparisons when using Scheffe test.
Where, The semen production trait (expressed in percentages) such as initial sperm motility was adjusted after angular transformation of the percentages as per Snedecor and Cochran (1987). While expressing the means and standard errors, angles were reconverted to percentages to a precision of two decimals.

TS = Test of signi cance
Statistical model is designed to estimate least squares means of semen production traits for the random effect of bulls and xed effect of non-genetic factors i.e. farm, ejaculate number, season of birth, period of birth, season of semen collection and period of semen collection Yrahbcfgx = μ + Rr + Sa + Zh + Tb + Uc + Xf + Yg + erahbcfgx

Results And Discussion
The least squares means (LSMs) for semen production traits i.e. semen volume, sperm concentration, initial sperm motility, post-thaw sperm motility and number of semen doses per ejaculate with the random effect of bull and xed effects of non-genetic factors such as farm, ejaculate number, season of birth, period of birth, season of semen collection and period of semen collection are given in Table 2. The results of type-3 tests of non-genetic factors and their interactions are given in Table 3.  Overall LSMs of semen volume per ejaculate was found to be 3.34 ± 0.18 ml in the present study, which was higher as compared to semen volume reports of 2. The semen collected in the rst ejaculate number gave signi cantly (P ≤ 0.01) higher semen volume (3.91 ± 0.18 ml) compared to second ejaculate number (2.77 ± 0.18 ml). Signi cantly higher semen volume was produced in the rst ejaculate collection in the present study. Lower semen volume in the second ejaculation was due to physiological effect which was always bound to occur in subsequent collection after rst collection. Similar ndings were also reported by Ramajayan (2016)  The semen collected in the rst ejaculation gave signi cantly (P ≤ 0.01) higher sperm concentration (1473.28 ± 75.62 million per ml) as compare to the second ejaculation (1003.11 ± 75.65 million per ml). Sperm concentration was signi cantly affected by ejaculate number and signi cantly higher sperm concentration was found in the rst ejaculate number in the present study. Similar ndings were also reported by Ramajayan (2016) in Murrah and Bhave et al. (2020) in pooled data of Banni, Bhadawari, Jaffarabadi, Murrah, Pandharpuri and Surti buffaloes.
Overall LSMs of initial sperm motility (70.55 ± 0.12 %) in the present study was higher as compared to initial sperm motility reports of 68.40 ± 1. The initial sperm motility was signi cantly (P ≤ 0.01) higher in farm-1 (70.96 ± 0.16 %) as compare to farm-2 (70.14 ± 0.12 %). Effect of period of birth on the initial sperm motility was found highly signi cant (P ≤ 0.01) in the present study which might be due to changes in the environmental condition and managemental practices over the periods. Effect of age of bull at rst semen collection on initial sperm motility was signi cant (P < 0.05) in the present study. Higher initial sperm motility of 70.61 ± 0.36 to 70.73 ± 0.07 % were found in the bulls with 1 to 3 years of age at rst semen collection which was at par with initial sperm motility found in the semen of 5 to 7 years of age of bull at rst semen collection. Highly signi cant (P ≤ 0.01) effect of period of semen collection was found on initial sperm motility. Highest initial sperm motility of 70.69 ± 0.12 % was found in the semen collected during the period of 2019 to 2020 whereas lowest initial sperm motility of 70.44 ± 0.12 % was found in the semen collected during the 2013 to 2014. It revealed that change in managemental and environmental condition during semen collection may contribute to differential initial sperm motility.
The semen collected in the second ejaculation gave signi cantly (P ≤ 0.01) higher initial sperm motility of 70.58 ± 0.12 % as compare to the rst ejaculation (70.53 ± 0.12 %). Lower initial sperm motility in the rst ejaculation was due to physiological effect which was always bound to occur as the rst ejaculation contains more non-viable sperms. Similar to the present study, Ramajayan (2016) in Murrah and Bhave et al. (2020) in pooled data of Banni, Bhadawari, Jaffarabadi, Murrah, Pandharpuri and Surti buffaloes also reported signi cant effect of ejaculate number on initial sperm motility.
Overall LSMs of post-thaw sperm motility was found to be 60.82 ± 0.16 % in the present study, which was higher as compare to postthaw sperm motility reports The post-thaw sperm motility was signi cantly (P ≤ 0.01) higher in farm-2 (70.17 ± 0.16 %) as compare to farm-1 (51.48 ± 0.18 %).
Effect of season of birth was signi cant (P ≤ 0.05) on post-thaw sperm motility. Post-thaw sperm motility of 60.96 ± 0.16 % was observed in the winter born bulls which signi cantly differed with monsoon born bulls' post-thaw sperm motility (60.73 ± 0.16 %).
Effect of period of birth was highly signi cant (P ≤ 0.01) on post-thaw sperm motility. Higher post-thaw sperm motility of 62.02 ± 0.22 % was observed in the 2016 to 2017 born bulls but it was lower (60.34 ± 0.21 %) in 2008 to 2009 born bulls which was at par with 2004 to 2007 and 2010 to 2013 born bulls. Effect of period of birth on the post-thaw sperm motility was found highly signi cant (P ≤ 0.01) in the present study which might be due to changes in the environmental conditions and managemental practices developed and adopted over the periods. Younger bulls produced semen with higher post-thaw sperm motility as compare to adult and older bulls in the present study.
Post-thaw sperm motility was not affected signi cantly by season of semen collection in the present study. Contrarily to the present study signi cant effect of season of semen collection on post-thaw sperm motility was reported by Bhave et al. (2020) in the pooled data of Banni, Bhadawari, Jaffarabadi, Murrah, Pandharpuri and Surti buffaloes.
Highly signi cant (P ≤ 0.01) effect of period of semen collection was found on post-thaw sperm motility. Post-thaw sperm motility of 61.04 ± 0.16 % was found to be signi cantly highest in the semen collected during the period of 2019 to 2020 as compare to postthaw sperm motilities during periods 1 to 4. Post-thaw sperm motilities of semen collected during period 1 to 4 were at par with each other. As the period of semen collection advanced post-thaw sperm motility increased showing better handling practices adopted by the semen stations with time.
The semen collected in the second ejaculation gave signi cantly (P ≤ 0.01) higher post-thaw sperm motility of 60.89 ± 0.16 % as compare to the rst ejaculation (60.75 ± 0.16 %). Post-thaw sperm motility was signi cantly affected by ejaculate number and signi cantly higher post-thaw sperm motility was found in the second ejaculation in the present study. Lower post-thaw sperm motility in the rst ejaculation was due to physiological effect which was always bound to occur as the rst ejaculation contains more nonmotile sperms due to gap between semen collection days and sperm production cycle in the reproductive organ as sperms get produce, mature and become non-motile till next semen collection. Similarly signi cant effect of ejaculate number on post-thaw sperm motility were reported by Ramajayan (2016)  Number of semen doses per ejaculate did not differ signi cantly between farm-1 and 2. Semen production traits like semen volume, sperm concentration, initial sperm motility and post-thaw sperm motility under present study were signi cantly differed between farm-1 and 2 but number of semen doses per ejaculate was not affected. Number of semen doses per ejaculate mainly depend on semen volume and sperm concentration. From the data of the present study it was observed that farm-1 has lower semen volume compared to farm-2 but sperm concentration was higher in farm-1 as compare to farm-2 which might have resulted in overall at par production of semen doses per ejaculate.
Effect of season of birth was non-signi cant (P > 0.05) on number of semen doses per ejaculate. This indicate overall adoption of bulls to the particular environment to achieve pubertal age and have well developed reproductive organs without in uence of seasonal variation. Number of semen doses per ejaculate was not affected signi cantly by period of birth of bull.
Number of semen doses per ejaculate were signi cantly (P ≤ 0.01) affected by season of semen collection. Semen collected in the monsoon season has higher number of semen doses per ejaculate (186.89 ± 11.85) which was followed by 185.23 ± 11.87 in summer, however difference between both of them was non-signi cant. Signi cantly lower number of semen doses was observed in the winter season of semen collection (172.69 ± 11.85). Number of semen doses per ejaculate were signi cantly affected by season of semen collection in the present study. Signi cantly higher number of semen doses per ejaculate were produced from the semen collected during summer and monsoon seasons compared to winter season. Semen characteristics like semen volume and sperm concentration were higher during the summer and monsoon seasons' collected semen. Hence, the higher number of semen doses per ejaculate were produced during summer and monsoon season of semen collection. Contrary to the present nding, Bhosrekar (1988) reported highest frozen semen doses in the winter season but he also narrated that rainy season seemed to be better for semen freezability and lower discard rate. Similar to the present study signi cant effect of season of semen collection on number of semen doses per ejaculate was also reported by Bhosrekar (1988) and Bhosrekar et al. (1992) in Surti buffalo.
Highly signi cant (P ≤ 0.01) effect of period of semen collection was found on number of semen doses per ejaculate. Higher number of semen doses per ejaculate (196.26 ± 11.86) was observed from the semen collected during 2015 to 2016 whereas lowest number of semen doses per ejaculate of 151.51 ± 12.32 was produced from the semen collected during 2011 to 2012. Signi cant (P ≤ 0.01) effect of period of semen collection on the number of semen doses per ejaculate was found in the present study. Relatively higher number of semen doses per ejaculate produced during 2015 to 2016 and 2019 to 2020 periods which might be due to better environmental and managemental practices during the periods.
The semen collected in the rst ejaculation produced signi cantly (P ≤ 0.01) higher number of semen doses per ejaculate (248.15 ± 11.83) as compare to the second ejaculation (115.06 ± 11.84).
In conclusion, Monsoon and summer were favorable seasons for semen collection because of higher sperm concentration which resulted in to higher semen doses per ejaculate in Mehsana buffalo bull. Additionally, Monsoon collected semen had highest volume. Hence, monsoon followed by summer season would be the favorable season for semen collection. Mature Mehsana buffalo bulls of 3 to 5 years of age or bulls having more than 700 kg body weight or the bulls, where semen collection was done after 2012 produced higher semen volume leading to higher semen doses per ejaculate. This indicates that bulls maturing at the age of 3 years (approx.) or having body weight of 700 kg or more produced more semen. First ejaculation had higher semen volume irrespective of age and season of semen collection resulting in to more semen doses per ejaculate in Mehsana buffalo bulls. Figure 1 Average monthly high and low temperature at PSK, Jagudan during the period of study Average monthly high and low temperature at DSPU, Dama during the period of study