<div style="text-align: justify;">The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphism in the GnRHR gene in Murrah bulls. Blood samples were collected from 109 Murrah bulls maintained at three different organized semen stations viz., Centralized Semen Collection Centre of Livestock Breeding and Training Centre, Dharwad; Nandini Sperm Station and State Livestock Breeding and Training Centre, Hessarghatta, Bengaluru, Karnataka, India. Genomic DNA was isolated from the blood samples within 24 hours of collection . Mutations were screened using Polymerase Chain Reaction &ndash; Single Stranded Conformational Polymorphism (PCR-SSCP) technique followed by Sanger Sequencing. PCR-SSCP method revealed similar band pattern within Murrah bulls. To confirm monomorphism in the studied population with respect to GnRHR gene, duplicate samples from each primer fragment were custom sequenced. PCR-SSCP and sequence analysis revealed monomorphism within the studied population that is coding region as well as exon-intron boundaries of GnRHR gene is highly conserved among Murrah bulls. However, a total of 28 Single Nucleotide variations (SNV&rsquo;s) have been found when compared with Bos Taurus reference sequence for GnRHR gene.</div>

Identification of Single Nucleotide Polymorphism in<em> GnRHR</em> gene in Murrah bulls

<p><span style="background-color:rgb(252, 252, 252); color:rgb(96, 96, 96); font-family:open sans; font-size:15px">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. </span></p>


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


<div><strong>Background: </strong>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 <em>DDX3Y</em> gene in Murrah buffalo bulls.&nbsp;<br />
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<strong>Methods: </strong>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 <em>DDX3Y</em> 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 <em>Bos taurus</em> sequence were determined using Clustal W. The phylogenetic tree and genetic distance were constructed using the MegAlign module.&nbsp;<br />
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<strong>Result: </strong>The analysis of sequences revealed that the exons and their adjacent intronic regions of the <em>DDX3Y</em> 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 <em>Bos taurus</em> 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 <em>DDX3Y</em> gene in a more extensive and diverse population of Murrah buffalo bulls distributed in different regions could aid to discover substantial SNPs.</div>


<p>Identification of Single Nucleotide Polymorphism in Y-chromosome Specific <em>DDX3Y </em>Gene in Murrah Buffalo Bulls</p>


<div style="text-align: justify;">The effect of dietary supplementation of Cu and Zn on semen quality parameters and certain bio-chemicals parameters were evaluated in Murrah bulls. Twelve mature Murrah bulls (4-6 years of age) were divided into three groups (n=4) T1, T2, and T3 based on semen volume and concentration and were fed as per ICAR standard (2013). However, the animals were supplemented with 0%, 25%, and 50% Cu and Zn above the basal diet in T1, T2 and T3 groups, respectively for 180 days. Quantitative and qualitative characteristics of semen, blood and seminal plasma antioxidant status, blood and semen minerals (Cu, Zn, Ca and Mn) were determined in experimental Murrah bulls. Semen ejaculate volume (mL) increased in T2 and T3 while sperm concentration (million/mL), intact acrosome (%), HOST reacted spermatozoa (%) increased in T3 group compared to T1 and T2 groups. No difference was observed in mass motility, pH, live spermatozoa (%) in semen sexual behaviour except dismounting time which was decreased in T3 than T1 and T2 (P&lt;0.05). SOD activity in blood plasma and LPO activity decreased in seminal plasma in both T2 and T3 groups than T1, whereas catalase activity did not show any significant difference. Cu and Zn supplementation in T2 group improved (P&lt;0.05) Zn level in blood and seminal plasma and Cu level in blood plasma only, but not in seminal plasma. Therefore, it can be concluded that supplementation of Zn and Cu at 50% above the recommended levels of ICAR (2013) improved the qualitative and quantitative attributes of semen in Murrah bulls.</div>

<div style="text-align: justify;">Effect of Dietary Copper and Zinc Supplementation on Semen Quality of Murrah Bulls</div>

<div style="text-align:justify">The present study was carried out with the objective to explore genetic polymorphism in Protein Phosphatase 1 Regulatory subunit 11 (PPP1R11) gene for its possible association with conception rate in Murrah bulls. Genomic DNA was isolated by phenol-chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-NDRI, Karnal. The quality and concentration of DNA was checked through spectrophotometer reading. The target region of PPP1R11 gene was amplified using 3 sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced, edited and aligned using Clustal Omega with Bubalus bubalis for identification of SNPs. A total of 7 SNPs were found, out of which 1 at exon 3 and 6 at 3&rsquo;UTR. SNP in exon 3 at nt.G1932C resulted in non synonymous (Serine to Threonine) type of amino acid substitution at position 109 of PPP1R11 gene in Murrah. PCR-RFLP was carried out using restriction enzyme BsmAI for genotyping the animals. All Murrah animals included in present study were monomorphic with CC genotype for the targeted region of PPP1R11 gene.&nbsp;</div>

Exploration of genetic polymorphism in targeted region of Protein Phosphatase 1 Regulatory subunit 11 (PPP1R11) in Murrah bulls

<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>

<div>An investigation was conducted to detect single nucleotide polymorphism in Inhibin alpha gene (INHA) and to analyse the association between the observed polymorphisms with semen quality traits in Murrah buffalo bulls. Genomic DNA was isolated from blood samples of 103 bulls, the amplified PCR products were further subjected to polyacrylamide gel electrophoresis. Band patterns were characterised by the number of bands, mobility shifts and scored manually. Representative PCR products giving unique SSCP patterns were custom sequenced using automated ABI DNA Sequencer to confirm the mobility shift in each pattern. Sequence data were analysed using DNA Baser and Bio edit software Clustal omega multiple alignments for detecting single nucleotide polymorphisms in comparison to observed sequence with the Bubalus sbubalis INHA gene reference sequence. INHA gene, exon1 was monomorphic, while exon 2 was found to be polymorphic. For simple interpretation of SSCP patterns, INHA exon 2 was divided into two fragments. The first fragment of INHA exon 2 (INHA exon 2 FR1) revealed a transition at the C267119T position and a transversion at the G267033T position in the coding region in comparison to Bubalus bubalis reference sequence. The second fragment of INHA exon 2 (INHA exon 2 FR2) exhibited one transition at T266510C position in comparison to the Bubalus bubalis reference sequence. The SSCP variants of the first fragment of INHA exon2 had a significant effect on semen volume per ejaculate (P&lt;0.05) and sperm concentration (P&lt;0.01). The SSCP variants of the second fragment of INHA exon 2 had a significant effect on sperm concentration (P&lt;0.01) and functional membrane integrity (P&lt;0.05). The observed association between SSCP variants in INHA gene with semen volume per ejaculate, sperm concentration and functional membrane integrity suggests the possibility of using these genetic variants in INHA gene as prognostic markers for selecting Murrah bulls with high semen quality traits.</div>


<div>Polymorphism in inhibin alpha gene and its association with semen quality traits in Murrah bulls</div>


<div><strong>Background:</strong> The beta-defensin family antigens present on sperm surface suggestively play pivotal role in sperm fertility by aiding in various steps of sperm maturation, motility, capacitation, immune recognition and sperm-oocyte interaction in female tract. It is imperative to explore genetic polymorphisms to build a better understanding of individual variation in male fertility. The experiment was designed to identify DNA polymorphism in Sperm Associated Antigen 11B (SPAG11B) gene and to analyze association between genetic variants with sperm quality traits in buffalo bulls (Murrah) in ICAR-National Dairy Research Institute, Karnal.&nbsp;<br />
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<strong>Methods: </strong>Genomic DNA was extracted from hundred and thirty Murrah bulls. A 395 base pair region covering partial Intron 2, Exon 3 and Partial Intron 3 of bovine SPAG11 gene was amplified and genotyped using PCR-RFLP method. The PCR amplified product was purified, sequenced and further ClustalW analysis was done to align edited sequence with reported Bos taurus sequence (AC_000158.1). Gene and genotype frequencies, effective number of alleles, level of heterozygosity and polymorphic information content of various genotypes were estimated by standard procedure. Seminal parameters (Post thaw sperm motility, sperm viability, acrosomal integrity, HOST and abnormality) were estimated and statistical analysis was carried out.&nbsp;<br />
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<strong>Result:</strong> A novel SNP (G&gt;A substitution) at 2266 base of the SPAG11B gene was identified by sequencing. The fragment of SPAG11 gene containing 395 base pairs was amplified using PCR and digested with restriction enzyme <em>i.e.</em> MunI which showed three distinct genotypes <em>viz.</em>, AA (266 bp and 107 bp fragment), AG (307 bp, 266 bp and 107 bp) and GG (373 bp fragment). Least squares means of seminal parameters for the SNP was estimated and compared after correction for non-genetic factors. Between genotypes, significant differences were found only for acrosomal integrity and was highest in AG (74.22&plusmn;0.72) followed by AA (72.6&plusmn;0.89) and GG (71.12&plusmn;0.97), respectively. The identified novel SNP of SPAG11 gene showed significant association with acrosome integrity with genotype AG being superior to other genotypes. However, an association cannot be established with other seminal parameters with this SNP, further studies are required in order to validate the impact of g.2266G&gt;A on sperm quality traits in a large population.</div>


<p>SNP Identification in Sperm Associated Antigen 11B Gene and Its Association with Sperm Quality Traits in Murrah Bulls</p>


<p><strong>Background:</strong> 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;<br />
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<strong>Methods:</strong> 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;<br />
<br />
<strong>Result:</strong> 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.</p>


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


<div><strong>Background:</strong> In a herd going through improvement by selective breeding, bulls contribute more than half of the genetic gain due to higher selection intensity and wider coverage through AI. On the other side, meritorious sires might have a lower fertility due to various reasons which would render them less fit in their contribution to genetic improvement. Thus dairy buffalo breeders must find a delicate balance between production and reproduction superiority for selection of sires, for which an accurate assessment of genetic and non-genetic component of conception is essential.&nbsp;<br />
<br />
<strong>Methods:</strong> Present study was conducted on thirty five years data comprising of 5349 AI records on 1302 Murrah buffaloes using 86 bulls maintained and semen analysis of cryopreserved semen samples of 130 Murrah maintained at ICAR-National Dairy Research Institute Karnal were analyzed to study bull conception rate and sperm quality traits. Data were classified into four periods; four seasons of AI viz., Summer (April-June), rainy (July-September), autumn (October-November), winter (December-March); five parity (first, second, third, fourth, fifth and above) and five female AI number (1,2,3,4 and &sup3; 5 AI) to study the conception rate. Age at freezing (Years), period of freezing and season of freezing were analysed to observe their effects on semen quality traits. Mixed model analysis (SAS 9.3) and the analysis of variance were used to see the effects of various factors on conception rate as well as on semen quality parameters.<br />
<br />
<strong>Result:</strong> The analysis of variance revealed that period of AI and season of AI had significant (P&lt;0.05) effect on conception rate while parity and female AI number were not found to affect the conception rate of bulls. For seminal parameters, acrosome integrity and HOST were significantly affected by age at freezing. Season affected all the semen quality traits while the effect of period of freezing was found to be non-significant for all the traits. Over all autumn season was found to be most suitable with respect to better seminal parameters. Considering season of AI, rainy season (July-September) had the lowest conception rate. The overall conception rate (%) in the study was 38.43&plusmn;0.99; where Sires showed a wide variation for conception rate ranging from 20.00% to 60.46%. Concluding with the fact that there exists variation among bull fertility that can be utilized for not only herd conception rates but also be used as a criteria for bull selection and evaluation.</div>


Identification of Single Nucleotide Polymorphism in GnRHR gene in Murrah bulls

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

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

Effect of Dietary Copper and Zinc Supplementation on Semen Quality of Murrah Bulls

Exploration of genetic polymorphism in targeted region of Protein Phosphatase 1 Regulatory subunit 11 (PPP1R11) in Murrah bulls

Digital infrared thermography as a tool to assess the effect of season, age and housing on seminal traits in Murrah bulls

Polymorphism in inhibin alpha gene and its association with semen quality traits in Murrah bulls

SNP Identification in Sperm Associated Antigen 11B Gene and Its Association with Sperm Quality Traits in Murrah Bulls

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

Effects of Various Non-genetic Factors on Fertility Traits in Murrah Bulls in an Organized Herd