<div>Background: Molecular markers based approaches are essential to select fertile bulls for frozen semen production at an early age. The present investigation was undertaken to perform the molecular characterization and identify single nucleotide polymorphisms (SNP) in Y-chromosome specific DDX3Y gene in Murrah buffalo bulls.&nbsp; 
 
Methods: The genomic DNA isolated from the blood samples of 70 Murrah buffalo bulls, covering bulls with normal seminal traits and poor production performance (poor semen quality, freezability, libido), were subjected to PCR amplification. The sequences of DDX3Y gene were analyzed for single nucleotide polymorphism using the seqman module of DNASTAR LASERGENE software. The single nucleotide variations in the sequences with reference to the Bos taurus sequence were determined using Clustal W. The phylogenetic tree and genetic distance were constructed using the MegAlign module.&nbsp; 
 
Result: The analysis of sequences revealed that the exons and their adjacent intronic regions of the DDX3Y gene are monomorphic in nature without any variations indicating that the sequences are highly conserved in the studied population of Murrah buffalo bulls. However, a considerable number of single nucleotide variations were observed in the sequences of Murrah buffalo compared with Bos taurus sequences. Furthermore, the phylogenetic tree analysis revealed less divergence and close genetic association between the sequences of Murrah buffalo and other species in the bovinae family than the caprinea species. Further studies on DDX3Y gene in a more extensive and diverse population of Murrah buffalo bulls distributed in different regions could aid to discover substantial SNPs.</div>

Identification of Single Nucleotide Polymorphism in Y-chromosome Specific DDX3Y Gene in Murrah Buffalo Bulls

Background: The aim of the study was to compare the effect of season on the semen quality traits of Murrah and Nili Ravi buffalo breeding bulls.&nbsp; 
 
Methods: For a period of 5 years, a total of 855 ejaculation records of 17 buffalo breeding bulls, including Murrah (11) and Nili Ravi (6) buffalo bulls maintained at an organised farm, were analysed for various semen traits using least squares analysis.&nbsp; 
 
Result: Breed had a significant effect (p&lt;0.01) on semen volume, consistency, mass activity (MA), total sperm count (TSC) and post thaw motility (PTM) and a non-significant effect on colour and initial motility (IM). In Murrah bulls, except for semen volume, the other parameters viz. consistency, MA, TSC and PTM were found to be significantly higher than Nili Ravi bulls. Season of semen collection had a significant effect on consistency, TSC and PTM whereas a non-significant effect on semen volume, colour, MA and IM was observed. During the summer season, overall consistency and TSC were higher, but PTM was found to be comparatively lower. Season and breed interaction had a significant effect (p&lt;0.01) on consistency, TSC and IM while a non-significant effect on semen volume, colour, MA and PTM was observed. In Murrah buffalo bulls, a non-significant effect of season on consistency was observed whereas, in Nili Ravi bulls, a significantly higher consistency during summer and spring and; a significantly lower consistency during the rainy season was observed. In Murrah bulls, the highest TSC were observed during the rainy and summer seasons; whereas, in Nili Ravi bulls only during the summer season. In Murrah bulls, semen consistency, MA and IM were lower during the spring season; semen volume was lower during the prewinter season; total sperm count was lower during the winter season. In Nili Ravi bulls, semen volume was lower during summer; consistency, MA and TSC were lower during the rainy season; and IM was lower during the spring season. The PTM was lower during the summer season in both the breeds.

A Comparative Study for Semen Quality Traits between Murrah and Nili Ravi Buffalo Breeding Bulls Maintained at an Organized Farm

<div style="text-align: justify;">Bull fertility is an important factor for genetic improvement; therefore prediction of fertility an early age of the bulls by using genetic markers is a goal for livestock breeding. Zinc finger protein280BY linked gene is predominantly expressed in testis and has a major role in spermatogenesis. An investigation was carried out to detect the genetic variants in the male specific region (MSY) of ZNF280BY gene and their association with semen quality traits in Murrah buffaloes. Polymerase Chain Reaction-Single Strand Conformation Polymorphism technique (PCR-SSCP) and DNA sequencing methods revealed three different SSCP band patterns (AA, BB, CC); one transition (C10375T) &nbsp;and an indel (insertion of T at 10460-10461). The associations of the genetic variants with semen volume, sperm concentration, Hypo Osmotic Swelling Test (HOST) in fresh and frozen semen were observed. AA and CC genotype bulls had high volume (P&pound;0.05) of semen per ejaculation than BB genotype bulls. BB genotype bulls had high sperm concentration (106/mL) per ejaculate in comparison to AA and CC genotype bulls (P&pound;0.05). AA and BB genotype bulls had a higher per cent of HOST (P&pound;0.05) reacted sperms of fresh semen and per cent acrosomal integrity of frozen semen in comparison to CC genotype bulls. The observed association between SSCP variants in MSY region of ZNF280BY gene with semen quality parameters indicated the possibilities of using ZNF280BY as a candidate gene for identification of markers for semen quality traits in Murrah buffaloes.</div>

<div style="text-align: justify;">Genetic variants in male specific region (MSY) of ZNF280BY gene and their association with semen quality traits in Murrah buffalo bulls</div>

<div>Background: Palm kernel cake is a high-fibre and medium-grade protein feed available at a cheaper price. Determining the optimum quantity of palm kernel cake inclusion levels could help in designing sustainable livestock-feeding systems. The studies related to its use in buffaloes are scarce. Considering the indispensable role of buffaloes in enhancing the Indian economy, the current study is aimed at finding the optimum level of palm kernel cake inclusion in rations of buffaloes. 
 
Methods: In a 4&times;4 LSD, four graded Murrah buffalo bulls (4 yrs; 329&plusmn;10.4 kg) were fed maintenance rations comprising Super Napier and concentrate mixture. A concentrate mixture with 20% CP was prepared using conventional feed ingredients and is used as control. De-oiled palm kernel cake (DPKC) is incorporated at 0, 15, 30 and 45 per cent level to study the effect of feeding DPKC on nutrient utilization, mineral (calcium, phosphorus and nitrogen) balance and rumen fluid parameters (TVFA and nitrogen dynamics). 
 
Result: The DPKC diets did not affect nutrient intakes; however, decreased the nutrient digestibility coefficients. The nitrogen outgo was higher and the N retention and per cent absorbed was lower for DPKC-included diets. No effects were observed on the calcium and phosphorus balance of diets. A linearly decreased pattern of TVFA and total nitrogen was observed with increased levels of DPKC. Further, the diets did not affect food and protozoal N and residual N of rumen liquor.</div>

Effect of Feeding Deoiled Palm Kernel Cake on Nutrient Utilization, Mineral Balance and Rumen Nitrogen Dynamics of Murrah Buffalo Bulls

Background: The integrity of storage tanks is essential in maintaining the liquid nitrogen used to preserve buffalo semen straws later utilized for artificial insemination. Faulty semen handling procedures, and equipment maintenance damage the spermatozoa leading to low-quality semen and low conception rates. 
&nbsp; 
Methods: The study used two brands of liquid nitrogen tanks and four purebred mature Bulgarian Murrah buffaloes. The semen samples were collected twice a week and immediately processed by the center&rsquo;s laboratory technician following a slow freezing technique. After processing, the semen straws were stored in LN2&nbsp;tanks at -196oC. 
&nbsp; 
Result: Results revealed that tank 2 was more efficient in maintaining the weekly amount of LN2&nbsp;and the frequent opening of tank covers significantly (P&lt;0.05) decreased the quantity of LN2&nbsp;after four days. Moreover, the different brands of LN2&nbsp;tanks did not affect sperm motility and livability, however, the frequency of exposing the semen straws to ambient temperature for 5 and 10 seconds lowered the sperm quality. In conclusion, semen straws should not be exposed to ambient temperature beyond the recommended time due to its deleterious effect on the semen quality compromising the success of insemination. 
&nbsp;

Effects of Storage Tanks and Ambient Temperature on the Liquid Nitrogen Evaporation and Semen Quality of Murrah Buffalo Bulls (Bubalus bubalis)

<div>Background: Majority of the people believed that, only the native breeds have A2 milk than exotic or crossbred animals. There is no enough research on milk protein variants have been carried out in Indian zebu cattle. Present study was conducted to screen more number of crossbred cattles and buffaloes in Tamil Nadu to identify the frequency of A1 and A2 alleles in the population. 
 
Methods: The study was conducted on 68 cattle and 172 murrah / graded murrah buffaloes to explore the polymorphic variants of &beta;-casein gene. Genomic DNA was extracted by phenol-chloroform method. Polymerase Chain Reaction (PCR) was performed with allele specific primers to amplify a 244 bp long fragment of beta-casein gene and visualized in 2% agarose gel electrophoresis. The population genetic indices were calculated based on the formulas. 
 
Result: In the present study revealed higher level of A2A2 genotype frequency (1.00) and fixation of A2 allele in kangayam cattle and murrah / graded murrah buffalo. The observed frequency of A1A1, A1A2 and A2A2 genotypes were 0.38,0.62 and 0.00 for jersey crossbred and 0.29, 0.71 and 0.00 for HF crossbred cattle. The range of Expected homozygosity (0.50 to 1.00), polymorphism information content (0.30 to 0.38), effective number of alleles (1.00 to 2.00) and level of possible variability realization value (44.54% to 100%) reflected existence of medium genetic variability in studied population.</div>

<div>A Study on &beta;-Casein Gene Polymorphism in Crossbred Cattle and Murrah / Graded Murrah Buffalo in Tamil Nadu</div>

<div>Background: Marker assisted selection based strategies are essential to identify superior quality bulls at an early age for frozen semen production. The present study aims to determine the genetic variants in the Y-chromosome specific genes, sex determining region on Y-chromosome (SRY) and testis specific protein Y-encoded (TSPY) in association with semen production traits in Murrah buffalo bulls.&nbsp; 
 
Methods: The genomic DNA extracted from the blood samples of 70 Murrah buffalo bulls maintained under semen production in the frozen semen station were utilized for PCR amplification. The sequences of SRY and TSPY genes were screened for single nucleotide polymorphism using the seqman module of DNASTAR LASERGENE software. The single nucleotide variations in comparison with reference sequences and phylogenetic tree construct were obtained using Clustal W and MegAlign module.&nbsp; 
 
Result: The analysis revealed that the sequences of SRY and TSPY genes were monomorphic in nature, devoid of variations specifying that the sequences are highly conserved among the Murrah buffalo bulls studied. Hence, association studies with seminal traits could not be performed. However, the exon sequence of SRY gene of Murrah buffalo showed a single nucleotide variation at position 209 (G&lt;A) compared to the sequences of Nili-Ravi and Kundi buffaloes. Similarly, the TSPY gene exhibited a significant number of single nucleotide variations compared with Bos taurus sequences. The phylogenetic tree analysis evinced a close genetic relationship with less divergence between Murrah buffalo and other buffalo breeds and species in the bovinae family. Further extensive studies on SRY, TSPY and other Y-chromosome specific genes in a large, diverse population of Murrah buffalo bulls could help to discover a considerable number of SNP markers related to semen production for the early selection of breeding bulls.</div>

Identification of Genetic Variants in Y-Chromosome Specific Genes Associated with Seminal Production in Murrah Buffaloes

In present investigation, the effect of non-genetic factors on age at first freezing and age at first use in Murrah breeding bulls has been studied. The data on reproduction traits of 57 Murrah bulls under NDRI (National Dairy Research Institute) centre belonging to 14 sets of Network Project on Buffalo Improvement at ICAR-NDRI, Karnal (Haryana), India during 20 years (1993-2013) were analysed using fixed linear model. The data were classified into various sub-classes for season of freezing and use, period of freezing and use, parity, stages of lactation and age groups of buffalo for age at first freezing and age at first use of Murrah breeding bulls. The average age at first freezing and use of Murrah bulls was estimated as 3.46 &plusmn; 0.08 years and 4.05 &plusmn; 0.13 years with the coefficient of variation of 14.43 % and 12.27%. The overall least-squares means for age at first freezing of Murrah bulls was estimated as 3.38 &plusmn; 0.01 years.

Effect of non-genetic factors on age at first freezing and age at first use in Murrah bulls

<div>Background: Bull fertility assumes a vital part in bovine reproduction. Since selection intensity of bulls picked for breeding programs in dairy industry is high as they can sire extensively larger counts of offspring as compared to their female counterparts. Therefore the present study was designed to establish the relationship between the sire fertility and performance traits of the Murrah buffaloes.&nbsp; 
 
Methods: A total of 12898 observations were recorded with respect to routine seminal parameters from 130 Murrah breeding bulls spanning between 1995 and 2019 (25 years). Data consisted of 2161 records of 305 days milk yield (305 MY) and lactation length (LL), 2160 records of lactation milk yield (LMY), 2155 records of peak milk yield (PMY), 1905 records of average fat percentage (FP), 1542 records of service period (SP) and 1914 records of conception rate (CR) have been utilized for the current study. Breeding values with respect to 33 Murrah buffalo bulls were taken for the correlation studied and for further ranking of the sires. 
 
Result: Average mean values of seminal parameter viz. ejaculated volume (EV), colour (Col), consistency (Con), log sperm concentration (LSC), mass activity (MA), initial motility (IM) and Post-Thaw Motility (PTM) were as 3.25&plusmn;0.01 (ml), 3.50&plusmn;0.01 (0-5 scale), 2.52&plusmn;0.04 (0-5 scale), 3.11&plusmn;0.01, 2.25&plusmn;0.01 (0-5 scale), 64.22&plusmn;0.15 and 38.62&plusmn;0.19 (%) respectively. The arithmetic means of LMY, 305 MY, PY, LL, FP, CR and SP were calculated to be 2343.33&plusmn;17.77, 2142.13&plusmn;13.77, 12.57&plusmn;0.06, 328.41&plusmn;2.11, 7.39&plusmn;0.01, 0.52&plusmn;0.09 and 166.36&plusmn;2.75, respectively. The sire rank correlation between the seminal attributes and performance traits was ranged between -0.57&plusmn;0.15 (ejaculate volume and lactation milk yield) and 0.49&plusmn;0.16 (semen consistency and lactation milk yield). Association between the fertility using routine semen quality parameters would be helpful to assess the bull fertility which lead to the improvement in the herd fertility.</div>

Genetic Study to Establish the Relationship between Sire Fertility and Daughter Performance Traits among Murrah Buffaloes

<div style="text-align:justify">The main objective of the current experiment was to assess the seasonal variation on seminal attributes in 109 Murrah buffalo (Bubalus bubalis) bulls using infrared thermography, maintained at commercial semen production stations with ideal nutrition and management conditions in Karnatka (south India). Data were collected on 6 ejaculates in three different seasons through &nbsp;a year with different age groups of bulls. Infrared thermograms and data were collected using an infrared FLIR i5 camera and analyzed by Quick report 1.2 SP2 softwere to determine the temperature gradient (TG, difference between the PPT and DPT) scrotal surface and Ocular temperature. Immediately after semen collection by AV method. Semen evaluation were performed twice per season throughout the year. The ejaculates were subjected to general linear model (GLM) analysis to determine the effect of various non-genetic factors viz., season (summer, rainy and winter), age (&lt;4, and &gt;4 year), housing (group and individual housing) on semen quality attributes of Murrah bull. The overall least square mean volume (VOL), sperm concentration (SCON&times;10v /ml), initial motility (IM), percent live spermatozoa (LIVE), acrosome integrity (AI), plasma membrane integrity (PMI), head abnormalities (HAN), midpiece abnormalities &nbsp;(MPAN), tail abnormalities (TAN) in fresh semen whereas post-thaw motility (PTM), acrosome integrity (AIPT) and plasma membrane integrity in post-thawed semen (PMIPT) were 3.88&plusmn;0.07 mL, 1041&plusmn;14.64 million/mL, 64.62&plusmn;0.50 %, 76.42&plusmn;0.51 %, 66.56&plusmn;0.48 %, 2.90&plusmn;0.05 %, 3.35&plusmn;0.05 %, 5.88&plusmn;0.10 %, 68.39&plusmn;0.47 %, 41.48&plusmn;0.55 % and 61.66&plusmn;0.47 %, respectively.The statistical analysis of variance revealed significant effect (P&lt;0.05) of season on semen quality attributes viz., VOL, SCON, IM, PTM, LIVE, HAN, MPAN, TAN, AI, AIPT, PMI and PMIPT. Age had significant effect on &nbsp;VOL, IM, LIVE, HAN, MPAN and &nbsp;house on SCON, PTM, AIPT and PMIPT.Thus, it may be concluded that the summer season adversely affected the various bio - physical characteristics of semen in Murrah buffalo bulls. Rainy season was the most favourable season for good quality semen production and the winter season might be considered as the intermediate between the two extremes.</div>

<div style="text-align:justify">Digital infrared thermography as a tool to assess the effect of season, age and housing on seminal traits in Murrah bulls</div>