Animal geneticists and breeders have the impending challenge of enhancing the resilience of Indian livestock to heat stress through better selection strategies. Climate change\'s impact on livestock is more intense in tropical countries like India where dairy cattle crossbreeds are more sensitive to heat stress. The main reason for this study was to find the missing relative changes in transcript levels in thermo-neutral and heat stress conditions in crossbred cattle through whole-transcriptome analysis of RNA-Seq data. Differentially expressed genes (DEGs) identified based on the minimum log twofold change value and false discovery rate 0.05 revealed 468 up-regulated genes and 2273 down-regulated significant genes. Functional annotation and pathway analysis of these significant DEGs were compared based on Gene Ontology (Biological process), Kyoto Encyclopedia of Genes and Genome (KEGG), and Reactome pathways using g: Profiler, ShinyGO v0.76, and iDEP.951 web tools. On finding network visualization, the most over-represented and correlated pathways were neuronal and sensory organ development, calcium signalling pathway, Mitogen-activated protein kinase (MAPK) and Smad signalling pathway, Ras-proximate-1, or Ras-related protein 1 (Rap 1) signalling pathway, apoptosis, and oxidative stress. Similarly, down-regulated genes were most expressed in mRNA processing, immune system, B-cell receptor signalling pathway, Nucleotide oligomerization domain (NOD)-like receptors (NLRs) signalling pathway and nonsense-mediated decay (NMD) pathway. The heat stress-responsive genes identified in this study will facilitate our understanding of the molecular basis for climate resilience and heat tolerance in Indian dairy crossbreeds.

OBJECTIVE: Karan Fries (KF), a high-producing composite cattle was developed through crossing indicine Tharparkar cows with taurine bulls (Holstein Friesian, Brown Swiss, and Jersey), to increase the milk yield across India. This composite cattle population must maintain sufficient genetic diversity for long-term development and breed improvement in the coming years. The level of linkage disequilibrium (LD) measures the influence of population genetic forces on the genomic structure and provides insights into the evolutionary history of populations, while the decay of LD is important in understanding the limits of genome-wide association studies for a population. Effective population size (Ne) which is genomically based on LD accumulated over the course of previous generations, is a valuable tool for e valuation of the genetic diversity and level of inbreeding. The present study was undertaken to understand KF population dynamics through the estimation of Ne and LD for the longterm sustainability of these breeds.
METHODS: The present study included 96 KF samples genotyped using Illumina HDBovine array to estimate the effective population and examine the LD pattern. The genotype data were also obtained for other crossbreds (Santa Gertrudis, Brangus, and Beefmaster) and Holstein Friesian cattle for comparison purposes.
RESULTS: The average LD between single nucleotide polymorphisms (SNPs) was r2 = 0.13 in the present study. LD decay (r2 = 0.2) was observed at 40 kb inter-marker distance, indicating a panel with 62,765 SNPs was sufficient for genomic breeding value estimation in KF cattle. The pedigree-based Ne of KF was determined to be 78, while the Ne estimates obtained using LD-based methods were 52 (SNeP) and 219 (genetic optimization for Ne estimation), respectively.
CONCLUSIONS: KF cattle have an Ne exceeding the FAO\'s minimum recommended level of 50, which was desirable. The study also revealed significant population dynamics of KF cattle and increased our understanding of devising suitable breeding strategies for longterm sustainable development.

Udder structure of milking cows is not merely vital to display the visual characteristics, but also important for high milk output and low mastitis risk incidence as well. The present study measured different udder type traits in Sahiwal (Bos indicus) and Karan Fries (Bos taurus × Bos indicus) and investigated their association with single nucleotide polymorphisms in Vitamin D Receptor and Protein Tyrosine Phosphatase, Receptor Type, R genes. GG genotype of SNP rs454303072 was found to have wider rear udder, larger udder circumference, longer distance between fore-rear teats and left-right teats in Karan Fries cattle. Whereas, in Sahiwal cattle, AA genotype of this SNP was found to be associated with the higher and wider rear udder, larger udder circumference and wider udder. AA genotype of SNP rs382671389 was found to be associated with longer fore teat in Karan Fries cattle. The TT and CC genotype of SNP rs435289107 was associated with udder type traits in Karan Fries and Sahiwal cattle respectively. These results suggest that BTA 5 harbors genomic regions associated with udder traits in Bos indicus and Bos indicus x Bos taurus cattle.

The present study was undertaken to explore the association of occurrence of clinical mastitis with udder and teat morphometry in cattle. The relationship of nine udder and eight teat morphometric traits with the occurrence of clinical mastitis was analysed using a binomial logistic regression model. A significant association of the udder and teat morphometric traits with the occurrence of mastitis was observed both in Karan Fries and Sahiwal cattle. The rear udder height, rear udder width, udder width and fore udder attachment were found to be the important udder morphometric traits associated with the occurrence of mastitis. In both groups, mastitic animals had broader rear udder width, lower rear udder height and loose fore udder attachment with lengthy and thicker teats. The inclusion of udder and teat morphometric traits in the selection and breeding program of Karan Fries and Sahiwal cattle may help in selection of animals for mastitis resistance.

The present study was conducted on Karan Fries cows to identify SNPs in breast cancer 1 (BRCA1) gene and their association with inflammation of mammary gland and production traits. Chi square analysis showed significant association of both SNPs i.e. G43737229T (P < 0.01) and G43761121A (P < 0.05) with incidence of clinical mastitis. Logistic modeling showed that GG genotype cows of G43737229T SNP was less susceptible to mastitis in comparison to TT genotype cows as the odds ratio of TT against GG for the non-incidence of mastitis was less i.e. 0.23. Whereas, cows with genotype AA of G43761121A SNP were less prone to clinical mastitis in comparison to GG genotype cows. G-T haplotype and GGTT combined genotype exhibited maximum mastitis incidences. Least squares analysis revealed significant association of G43737229T SNP with total milk yield (P < 0.01), 305 days milk yield (P < 0.01) and lactation length (P < 0.05). The TT and GT genotyped Karan Fries cows were associated with higher milk yields during first and second lactation, as well as with first lactation length. These observations on SNPs, genotypes and combined genotypes of BRCA1 gene and their differential association with the incidence of mastitis and production traits can be utilized as an aid to selection for simultaneous improvement of both antagonistic traits.

OBJECTIVE: The objective of this study was to evaluate the effects of protected fat plus yeast, niacin, zinc, and chromium dietary supplementation on the reduction of heat stress in Karan Fries (KF) heifers during hot humid months.
METHODS: The basal ration for both the control and treatment groups was the same, containing maize as green fodder and concentrate mixture. However, the treatment group was supplemented with protected fat (2.5% of dry matter intake [DMI]), yeast (10 g/animal/day), niacin (6 g/animal/day), zinc (40 mg/kg DMI), and chromium (1.5 mg/kg DMI).
RESULTS: The overall mean value of afternoon rectal temperature for control and treatment group was 103.17±0.09 and 102.72±0.10°F, respectively, and was significantly (p<0.01) lower in the treatment group. The overall mean value of afternoon respiration rate for control and treatment group was 76.35±0.56 and 73.13±0.58 breaths/min, respectively, and was also significantly (p<0.01) lower in the treatment group. The overall mean value of afternoon pulse rate for control and treatment group was 97.09±0.63 and 94.67±0.67 beats/minute, respectively, and was also significantly (p<0.01) lower in the treatment group. Finally, the mean cortisol concentration for control and treatment group was 3.94±0.05 ng/ml and 3.70±0.06 ng/ml, respectively, and was significantly (p<0.01) lower in the treatment group.
CONCLUSIONS: The present study shows that supplementation with the above feed additives could serve as a heat stress abatement strategy in growing KF heifers during extreme conditions in summer months.

Skin is most important environmental interface providing a protective envelope to animals. It\'s always under the influence of both internal and external stressors. Heat shock proteins (HSP) are highly conserved stress proteins which play crucial roles in environmental stress tolerance and thermal adaptation. Present study was planned to observe the relative mRNA expression of inducible (HSP70.1 and HSP70.2) and constitutive (HSP70.8) HSP in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle during different seasons. Skin biopsies were collected from rump region of each animal, aseptically during winter, spring and summer season. Quantitative real time polymerase chain reaction was performed to examine the gene expression of constitutive (HSP70.8) and inducible (HSP70.1 and HSP70.2) HSP in skin of both the breeds during different seasons. Present study observed higher expression of both constitutive and inducible HSP genes in both the breeds during summer and winter than spring season, but magnitude of increase was higher during summer than winter. During summer season, expression pattern of HSPs in skin showed breed differences, where constitutive HSP expression was higher in Tharparkar than Karan Fries and that of inducible HSP was higher in Karan Fries than Tharparkar. Hence, present study suggested that HSP may be conveniently used as biomarkers for assessing protective response of skin against heat stress in zebu and crossbred cattle. Variation in expression between breeds is associated with their heat tolerance and thermal adaptability. In summary, skin of zebu cattle (Tharparkar) is more resistant to summer stress than crossbred (Karan Fries), providing greater protection against heat stress during summer season. Superior skin protective mechanism of zebu (Tharparkar) than crossbred (Karan-Fries) cattle against heat stress may contribute to superior adaptability of zebu cattle to tropical climatic conditions than crossbreed.

OBJECTIVE: The objective of this study was designed to estimate the metabolic heat production and methane emission in Sahiwal and Karan Fries (Holstein-Friesian X Tharparkar) heifers under two different feeding regimes, i.e., feeding regime-1 as per the National Research Council (NRC) (2001) and feeding regime-2 having 15% higher energy (supplementation of molasses) than NRC (2001).
METHODS: Six (n = 6) healthy heifers of Sahiwal and Karan Fries with 18-24 months of age were selected from Indian Council of Agricultural Research-National Dairy Research Institute, Karnal. An initial 15 days was maintained under feeding regime-1 and feeding regime-2 as adaptation period; actual experiment was conducted from 16(th) day onward for next 15 days. At the end of feeding regimes (on day 15(th) and 16(th)), expired air and volume were collected in Douglas bag for two consecutive days (morning [6:00 am] and evening [4:00 pm]). The fraction of methane and expired air volume were measured by methane analyzer and wet test meter, respectively. The oxygen consumption and carbon dioxide production were measured by iWorx LabScribe2.
RESULTS: The heat production (kcal/day) was significantly (p<0.05) higher in feeding regime-2 as compared to feeding regimen-1 in both breeds. The heat production per unit metabolic body weight was numerically higher in feeding regime-1 than feeding regime-2; however, the values were found statistically non-significant (p>0.05). The energy loss as methane (%) from total heat production was significantly (p<0.05) higher in feeding regime-1. The body weight (kg), metabolic body weight (W(0.75)), and basal metabolic rate (kcal/kg(0.75)) were significantly (p<0.05) higher in feeding regime-2 in both breeds.
CONCLUSIONS: This study indicates that higher energy diet by supplementing molasses may reduce energy loss as methane and enhance the growth of Sahiwal and Karan Fries heifers.

OBJECTIVE: The present study was carried out on semen ejaculates of 15 Karan Fries (KF) bulls maintained at Artificial Breeding Research Centre, National Dairy Research Institute, Karnal, India with an objective to evaluate the relationship between the conventional and fluorescent based semen quality analysis of the bulls.
METHODS: A total of 96 ejaculates were collected from 15 KF (Holstein Friesian [HF] crossbred) bulls. Semen were evaluated for color, volume, mass activity (MA) and percentage of individual motility (IM), sperm concentration, percent live spermatozoa, hypo-osmotic swelling test and acrosome integrity, chromatin integrity, sperm viability, and membrane integrity. Data were analyzed using SPSS software package for descriptive analysis. The correlation between rankings of sires based on conventional and fluorescent semen parameters were calculated by Spearman\'s rank correlation coefficient.
RESULTS: The average ejaculates volume (ml), sperm concentration (10(6)/ml), MA, IM (%), live (%), morphological abnormalities (%), host (%), acrosome integrity (%), chromomycin A3 (CMA3) (%), SYBR-PI (%), and fluorescent isothiocyanate-peanut agglutinin (FITC-PNA) (%) were 4.57±0.36, 1162.98±97.93, 2.95±0.09, 60.8±1.22, 71.41±2.10, 9.31±1.15, 65.5±1.81, 86.6±1.59, 3.53±0.43, 65.39±2.23 and 74.47±2.53, respectively. Rank correlations were found to be significant for SYBR-PI and FITC-PNA with most of the parameters evaluated by conventional methods. Overall, among conventional criteria, IM revealed ranking of bulls almost similar to that of fluorescent criteria.
CONCLUSIONS: Overview of our results indicated that, among conventional criteria, MA and IM revealed ranking of bulls almost similar to that of fluorescent criteria.

Heat shock proteins (HSPs) act as molecular chaperones those are preferentially transcribed in respose to heat stress and the polymorphism in HSP genes associated with heat tolerance traits in cows. HSP90AA1 gene has been mapped on Bos taurus autosome 21 (BTA-21) and spans nearly 5368 bp comprising of 11 exons out of which the first exon does not translate. The present study was done on Karan Fries (5/8 HF × 3/8 Tharparkar) cows reared in tropical climate with the objectives of identifying single-nucleotide polymorphisms (SNPs) in targeted regions (exon 3) of HSP90AA1 gene and analyzing their association with heat tolerance traits in Karan Fries cows. Respiration rate (RR) and rectal temperature (RT) were recorded once daily for four consecutive days during probable extreme hours in different seasons or temperature humidity index (THI), viz., winter, spring, and summer. For detecting single-nucleotide polymorphisms, sequence data were analyzed using BioEdit software (version 7.2). Comparative sequence analysis of HSP90AA1 gene showed point mutation, viz., g.1209A>G (exon 3) as compared to Bos taurus (NCBI Ref Seq: AC_000178.1). Association analysis indicated that THI was influenced (P < 0.01) by RR, RT, and HTC. Similarly, SNPs at locus g.1209A>G were categorized into three genotypes, i.e., AA, AG, and GG, and the least squares means (LSMEANS) of RR, RT, and HTC for GG (homozygous) genotype were significantly lower (P < 0.01) than AA (homozygous) and AG (heterozygous) genotypes. These findings may partly suggest that cows with GG genotypes were favored for heat tolerance trait, which can be used as an aid to selection for thermo-tolerance Karan Fries cows for better adaptation in subtropical and tropical hot climate.