Sperm cryopreservation is an important adjunct to assisted reproduction techniques (ART) for improving the reproductive efficiency of dairy cattle and buffaloes. Improved understanding of mechanisms and challenges of bovine semen cryopreservation is vital for artificial insemination on a commercial basis. Although cryopreservation of bovine spermatozoa is widely practiced and advanced beyond that of other species, there are still major gaps in the knowledge and technology. Upon cryopreservation, disruption of spermatozoal plasma membrane configuration due to alterations in metabolic pathways, enzymes and antioxidants activity add to lower efficiency with loss of sperm longevity and fertilising ability. Therefore, the effective amalgamation of cryo‐variables like ambient temperature, cooling and thawing rates, nucleation temperature, type and concentration of the cryoprotectant, seminal plasma composition, free radicals and antioxidant status are required to optimise cryopreservation. Novel strategies like supplementation of cholesterol‐loaded cyclodextrins (CLC), nanovesicles, osteopontin, antioxidants, etc., in an extender and recent techniques like nano‐purification and modified packaging have to be optimised to ameliorate the cryodamage. This article is intended to describe the basic facts about the sperm cryopreservation process in bovine and the associated biochemical, biophysical, ultra‐structural, molecular and functional alterations.

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Background: Cryopreservation is an invaluable technique yet it is also known to be detrimental to sperm function and fertility due to cryo-injury and concomitant generation of reactive oxidants. During laboratory manipulation for the cryopreservation and freeze-thaw process, spermatozoa undergo osmotic stress, ionic imbalance, metabolic decoupling, membrane phase transition, destabilization of the cytoskeleton and antioxidant depletion which communally hampers the semen quality.Methods: With the aim of determining implications of cryopreservation and storage, semen samples were collected by artificial vagina technique from 12 Murrah bulls and subsequently examined at 0 hour (before cryopreservation) and at 24 hour, 1 month and 2 month of storage for various seminal attributes. Simultaneously seminal plasma was separated and preserved at -20oC till the analysis of biochemical indicators of semen quality viz., nitric oxide (NO), total antioxidant quantity (TAC) and lipid peroxidation status (TBARS). Result: A sharp reduction (p less than 0.01) in the semen quality was observed only at 24 h after cryopreservation except for viability. Significant reduction (p less than 0.05) in viable counts was observed up to 1 month interval. The capacitated sperm percentage was greater (p less than 0.01) in the cryopreserved semen as compared to fresh ejaculate. The mean ± SE levels of NO (μmol/L), TAC and TBARS (Units/ml) was 2.31±0.27, 0.73±0.04 and 1.11±0.16 respectively in seminal plasma of neat semen stored at -20oC, while the values in the extended seminal plasma after cryopreservation was 2.37±0.31, 0.44±0.03 and 0.65±0.03 respectively. So it can be inferred that most of the damage encountered by spermatozoa is during the initial period of freezing, but the damage associated by various stressors cannot be ignored.

Breeding bull fertility is an economically important trait in dairy cattle production (Taylor, Schnabel, & Sutovsky, 2018). Subfertility/ infertility in breeding bulls is one of the major reasons behind reproductive failure in dairy cattle (DeJarnette et al., 2004). Reproduction inefficiency in bulls has a direct impact on the overall herd profitability by leading the system to reduced incomes (longer calving intervals, reduced milk yield and higher culling rates) (Seegers, Fourichon, Malher, & l'Hostis, 1994), and hence efforts are needed to improve fertility traits by selective breeding (Walsh, Williams, & Evans, 2011).Direct selection for the bull fertility traits is not effective due to its

Purpose: The objective of the present study is to identify important polymorphisms at single nucleotide in Holstein crossbred bull spermatozoa and to identify genetic variants signi cantly in uence or associated to bull fertility. Methods: Spermatozoa from high-(HF) and low-fertile (LF) breeding bulls were subjected to high throughput Next-generation sequencing to identify important SNPs and novel variants associated with fertility.Results: A total of 77,036 genome wide SNPs were identi ed in the crossbred bull spermatozoa with a minimum read depth of 20, and among them, 10,269 were observed to be novel variants. A total of 42,290 and 34,748 variants were recorded in HF and LF bulls respectively, among which 6115 and 4673 were found to novel in respective group. Higher number of SNPs and Indels were identi ed in HF compared to LF bulls. GO analysis of ltered genes with signi cant variation in HF bulls indicated their enrichment in various important pathways like oxidative phosphorylation and metabolic pathways. On the other hand, GO analysis of ltered genes with signi cant variation in LF bulls revealed their involvement in Ca 2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly.Conclusions: The study identi ed SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13 and RPS27A, which are linked to sperm functionality, survival, protection from oxidative stress and bull fertility. The identi ed SNPs could be used as a tool for identi cation and selection of bulls for high fertility upon validation in large number of bulls