High serum free fatty acids and low leptin levels: Plausible metabolic indicators of negative energy balance in early lactating Murrah buffaloes.

Lactation is a highly demanding event in mammals, including buffaloes. It modulates the partitioning of nutrients, energy utilization, and food intake of the mother to meet her own and infant's energy needs. Failure to satisfy these energy needs leads to Negative Energy Balance (NEB). Currently, the only available indirect NEB indicator is Body Condition Score (BCS). However, direct dependency of the BCS on the peak depletion of body fat causes its inefficient use in a dairy farm. Thus, to establish objective NEB indicators in buffaloes, the serum levels of biochemical (serum ß-hydroxybutyrate [BHBA] and free fatty acids [FFAs]), and endocrine (Growth Hormone [GH], insulin-like growth factor1 [IGF1], Insulin, and leptin) parameters were estimated in buffaloes. Our results revealed that serum FFA levels were significantly (p < 0.05) higher in high milk yielders (HMY) than low milk yielders (LMY) and heifers (H) during the 3rd and the 4th weeks of postpartum. The serum FFA levels were also significantly (p < 0.001) higher in the postpartum buffaloes with BCS < 3 in the field conditions. Further, serum leptin levels were significantly (p < 0.05) lower in HMY than LMY during the 3rd week of postpartum. However, the BHBA, GH, IGF1, and insulin levels were not significantly different between lactating buffaloes and H. These observations indicated that the NEB condition is probably restricted to the first month of early lactation in buffaloes. In conclusion, the simultaneous higher FFA and lower leptin levels could act as direct plausible metabolic indicators of NEB in buffaloes.

A synonymous single nucleotide polymorphism (g.36417726C > A) in the Lama2 gene influencing fat deposition is associated with post-partum anestrus interval in Murrah buffalo.

Postpartum absence of estrus exhibition known as postpartum anestrus interval (PPAI) for more than 90 days after calving is a concerning issue for dairy buffalo farmers' economy. The PPAI duration is influenced by both management practices and animal genetics. Investigating genetic markers associated with PPAI is crucial for incorporating them into marker-assisted selection programs. Towards this goal, our study focused on exploring potential genetic markers from early postpartum adipose tissue gene networks. We successfully identified 24 Single Nucleotide Polymorphisms (SNPs) within 9 candidate genes. In our initial analysis involving 100 buffaloes, we detected a significant association (P = 0.02267) between a specific synonymous SNP within the Lama2 gene (g.36417726C > A) and PPAI. This finding was subsequently validated (P = 0.02937) in a larger cohort of 415 buffaloes, where the SNP explained 1.36 % of the genetic variance. Intriguingly, buffaloes with the CC genotype of this SNP exhibited a PPAI that was 12.71 ± 3.21 days longer compared to buffaloes with AA and CA genotypes. To gain insight into the functional relevance of this SNP, a computational analysis was performed which indicated that the C allele of the SNP (g.36417726C > A) increased the stability of LAMA2 mRNA compared to the A allele. This computational prediction was corroborated by observing a significant increase (P = 0.01798) in Lama2 gene expression (greater than 8-fold) and higher fat percentage (P < 0.05) in adipose tissue of CC genotypes (48.78 ± 1.87 %) compared to AA genotypes (33.59 ± 4.5 %). Furthermore, we noted a significant (P < 0.05) upregulation of C/ebpß, Pparγ, Fasn, C/ebpα, and Pnpla2 genes, along with the downregulation of Bmp2 and Ptch1 in CC genotypes as opposed to AA genotypes. This observation suggests the involvement of the Pparγ-mediated pathway in both adipogenesis and lipolysis within CC genotypes. In summary, our comprehensive analysis involving association and functional validation underscores the potential of the SNP (g.36417726C > A) within the Lama2 gene as a promising genetic marker against extended PPAI in Murrah buffalo.

Meprin A1 subunit beta gene polymorphism is associated with the length of post-partum anestrus interval in Murrah buffaloes.

Postpartum anestrus interval (PPAI) is the interval between parturition and the first postpartum estrus exhibition in animals. Appearance of both normal and PPA buffaloes under the same farm conditions indicates the role of possible genetic predisposition to PPA. To identify the genetic and non-genetic factors associated with PPA in buffaloes, we collected data on PPAI and other 38 non-genetic variables from 575 Murrah buffaloes in the field conditions and identified the PPA associated non-genetic factors in our previous study. To explore the genetic factors associated with the unexplained variation in PPAI residuals, the present study identified 41 single nucleotide polymorphisms (SNPs) in 13 candidate genes using Sanger sequencing. Exploration of their association with the PPAI residuals of 50 extreme PPA and 50 normal buffaloes identified the significant (P < 0.01) association of the SNP (g.37219977A>G) in the 3'-UTR region of the Meprin A 1 subunit beta (Mep1b) gene with PPAI, which was further validated (P = 0.058) in a large population sample (n = 417). Bioinformatics analysis of the 3'-UTR region has identified three miRNA, bta-miR-2420, bta-miR-2325b and bta-miR-453 that could regulate Igf-1 in the plasma of animals with different genotypes (GG, AG and AA). The higher Igf-1 levels in the GG genotypes than that of AA and AG genotypes of this SNP (g.37219977A>G) further suggest the association of Mep1b gene with PPA condition in Murrah buffaloes. As a result of this study, we propose that buffaloes with protective alleles at this SNP be selected to improve the herd's reproductive efficiency.

A single nucleotide polymorphism of the thyrotropin releasing hormone degrading ectoenzyme (TRHDE) gene is associated with post-partum anestrus in Murrah buffalo.

Thyrotropin releasing hormone degrading enzyme (TRHDE) gene is implicated in Thyrotropin releasing hormone (TRH) mediated prolactin secretion. It has been shown that the prolactin secretion alters the Gonadotropin-releasinghormone(GnRH) mediated estrous cycle. Therefore, TRHDE may also regulate postpartum anestrus. Earlier studies reported the role of non-synonymous single nucleotide polymorphism (SNPs) in various pathophysiological conditions by altering the structure and function of the proteins. Hence, in the present study, we identified SNPs in the putative promoter, first exon, middle exon and 3'-UTR containing the last exon of TRHDE gene and determined their association with postpartum anestrus (PPA) in Murrah buffaloes. We found one non synonymous SNP (G > C at 118095875 bp on chromosome 4) in the first exon of TRHDE and performed its association analysis in a population sample of 50 extreme PPA (residual PPAI: 123.06 ± 12.98 days) and 50 normal (residual PPAI: -80.46 ± 3.19 days) buffaloes. The residual PPAI value was the observed PPAI adjusted for the effect of 38 non-genetic factors. The analysis showed a significant (P < 0.004167) association of this SNP with PPA in buffaloes. Molecular dynamics simulations (MDS) also supported that the C allele altering Glutamine to Histidine at the amino acid 148 of TRHDE could enhance the stability and rigidity of TRHDE protein, which may lower its activity, increase TRH and prolactin, and reduce GnRH in PPA buffaloes. The MDS analysis further strengthens the association of the SNP (G > C) in the TRHDE gene with PPA condition in Murrah buffaloes. However, further investigation is needed to prove the MDS observations.

Body condition score, parity, shelter cleanliness and male proximity: Highly associated non-genetic factors with post-partum anestrus in Murrah buffalo in field conditions.

Postpartum anestrus (PPA) is a major reproductive problem in buffalo. Although both genetic and non-genetic factors influence the incidence of PPA, identifying associated non-genetic factors would be helpful for effective management of this problem. No systematic study, however, has been conducted to identify the association of non-genetic factors with PPA in buffalo, especially in field conditions. Data for 39 variables affecting postpartum anestrus interval (PPAI), health, nutrition, management and environmental status of the animals were collected on 575 buffalo from 15 villages. Data were analyzed using a general linear model considering PPAI as a dependent variable and the remaining 38 non-genetic parameters as independent variables. The results from the analysis indicated body condition score (BCS) variables, such as pelvic girdle score (P <  0.0001) and girth (P <  0.05), parity (P < 0.0001), shelter cleanliness (P <  0.001), male proximity (P <  0.005), amount and frequency of concentrate feeding during the early postpartum period (P <  0.05), general feeding frequency in a day (P <  0.05) and the treatments with oxytocin at the time of milking to facilitate milk release from the udder (P <  0.05) were associated with PPAI in Murrah buffalo. Plotting the PPAI residuals indicated the possibility of other unexplained factors, including genetics, that could also affect the differences in values for this variables in Murrah buffalo. In conclusion, a greater understanding of associated non-genetic factors with PPAI would help farmers in conducting proper managemental practices for early induction of reproductive cycles following parturition in Murrah buffalo.