Comparative RNA isolation methods from fresh ejaculated spermatozoa in Sahiwal cattle (Bos indicus) and Murrah buffalo (Bubalus bubalis) bulls for high quality and enhanced RNA yield.

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. The aim of the study was to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bulls. Ten fresh ejaculates from each Sahiwal (n = 10 bulls × 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) resulted significantly (p < .05) higher total RNA quantity (300-340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm.

Salivary cell-free HSD17B1 and HSPA1A transcripts as potential biomarkers for estrus identification in buffaloes (Bubalus bubalis).

Estrus detection is a major problem in buffaloes because of the poor expression of estrus signs leading to low reproductive efficiency. Salivary transcripts analysis is a promising tool to identify biomarkers; therefore, the present study was carried out to evaluate their potential as estrus biomarkers. The levels of HSD17B1, INHBA, HSPA1A, TES transcripts were compared in saliva during estrous cycle stages [early proestrus (day -2, EP), late proestrus (day-1, LP), estrus (E), metestrus (ME) and diestrus (DE)] of cyclic heifers (n = 8) and pluriparous (n = 8) buffaloes by employing quantitative real-time polymerase chain reaction (qRT-PCR). The levels of HSD17B1 (EP/DE 1.46-2.43 fold, LP/DE 2.49-3.06 fold; E/DE 7.21-11.9-fold p < 0.01; ME/D 1.0-1.16 fold) and HSPA1A (EP/DE 0.93-2.39 fold, LP/DE 2.68-3.23 fold; E/DE 8.52-15.18 fold p < 0.01; ME/D 0.86-1.01 fold) were significantly altered during the estrus than other estrous cycle stages in both cyclic heifers and pluriparous buffaloes. Receiver operating characteristic curve analysis revealed the ability of salivary HSD17B1 (AUC 0.96; p < 0.001) and HSPA1A (AUC 0.99; p < 0.01) to differentiate E from other stages of the estrous cycle. Significantly higher levels of HSD17B1 and HSPA1A transcripts in saliva during the estrus phase suggest their biomarkers potential for estrus detection in buffaloes.

Salivary crystallization pattern: a possible unconventional tool for timing of insemination and early pregnancy diagnosis in zebu cows.

The present study assessed if salivary crystallization pattern (ferning pattern formed as a result of the higher levels of salt content in the dried sample) could be used for estrus detection and for diagnosis of pregnancy/non-pregnancy in dairy cows. Saliva and blood samples were collected from non-pregnant cycling cows (Sahiwal breed; n = 20) on alternate days from the day of estrus till next estrus. Then, all the cows were inseminated and saliva and blood sampling were continued further for a period of 22 d post-insemination. Pregnancy diagnosis was carried out on day 45 post-insemination and eight cows were found to be pregnant. The salivary crystallization pattern and estradiol:progesterone ratio during estrous cycle and during pregnancy were compared among these cows. Six types of salivary crystallization patterns were discerned; distinct patterns such as branch-like, fern-like, fir-like and combinations of these. Fern-like pattern was observed in all the cows on the day of estrus (first measurement day) and furthermore, all of the cows that subsequently became pregnant had fern-like salivary crystallization pattern at the time of insemination. Saliva of all the pregnant cows showed branch-fir type of crystallization pattern on day 16 post-breeding while only 50% of non-pregnant cows showed this pattern on day 16 of estrous cycle. The appearance of fern-like pattern was positively and significantly related to estradiol:progesterone ratio (r = 0.86; P < 0.001). The findings were validated on a separate group of cycling cows (n = 32). We can conclude that salivary crystallization pattern might serve as a non-invasive and cost effective and easy-to-use cow-side tool for estrus detection and early pregnancy/non-pregnancy diagnosis in cows upon validation on a larger sample size.

Moringa oleifera leaf meal supplementation improves nutrient digestibility, milk yield, and reproductive performances in dairy cows during early lactation.

The study aimed to evaluate the effects of Moringa oleifera leaf meal (MOLM) supplementation on nutrient utilization, milk yield, and reproductive performance of early lactating Sahiwal cows. Control cows (GC) received a basal diet, while the treatment cows (GM) were supplemented with concentrate comprising 12% MOLM. Ovarian activity and uterine involution were monitored by trans-rectal ultrasonography on the 21st, 28th, 35th, and 42nd days postpartum. The result indicated that MOLM-supplemented cows required fewer days (P ≤ 0.05) to complete uterine involution. As lactation progresses, there was a significant reduction (P ≤ 0.05) in the diameter of the cervix and uterine horns in GM than GC. There was a significant increase in the number of follicles on the 21st day and average milk yield in GM than GC. The incidence of endometritis and cystic ovarian disease was less in MOLM supplemented group. The use of MOLM in the diet reduced the total cost per cow per successful service. It is concluded that MOLM can be safely included at 12% in the diet of early lactating cows to modulate the reproductive performances of dairy cows. Dairy farmers can use moringa leaf meal to feed their dairy cows, which is cheaper and improves production and reproduction performance.

Precision technologies for the management of reproduction in dairy cows.

Precision livestock farming (PLF) utilizes information and communication technology (ICT) to continuously monitor, control, and enhance the productivity, reproduction, health, welfare, and environmental impact of livestock. Technological advancements have facilitated the seamless flow of information from animals to humans, enabling practical decision-making processes concerning health, reproduction management, and calving surveillance. With the increasing population of livestock per farm, it has become impractical for farmers to individually track every animal within these large groups. Historically, cattle management decisions heavily relied on human observation, judgment, and experience. However, it is impossible for a single individual to gather reliable audio-visual monitoring data round the clock. Presently, dairy cows exhibit subtler indicators of estrus, resulting in a substantial chance of missing an estrus cycle. Furthermore, calving complications sometimes go unnoticed on farms, resulting in a higher number of culled cattle. In addition, an increasing number of crossbred cows experience delayed return to estrus after calving due to low body condition scores (BCS). The decline in BCS during the dry period is associated with a reduced likelihood of pregnancy following the first and second postpartum inseminations. Precision technologies enable the monitoring and tracking of an individual cow's physiological behavior and reproductive parameters, thereby optimizing management practices and farm performance. Despite the exploration of various technologies, there are still some common challenges that need to be addressed, including battery lifespan, transmission range, specificity and sensitivity, storage capacity, and economic affordability. Nonetheless, the demand for these tools from farmers and researchers is growing, and the implementation of PLF in grazing systems can yield positive outcomes in terms of animal reproductive welfare and labor optimization. This review primarily focuses on the different aspects of reproduction management in dairy using sensors, automated cameras, and various computer software.

Effect of over dilution of semen with tris extender on motion and functional attributes of bull spermatozoa during cryopreservation.

The present study was aimed to evaluate the effect of over dilution of semen with tris extender on motion and functional attributes of bull sperm post-thaw. Ejaculates (n = 24; mass motility ≥3+) were collected from bulls (n = 4) using artificial vagina, diluted to 20, 15, 10 and 5 million spermatozoa/0.25 ml, and cryopreserved. The results revealed that total motility (%), progressive motility (%) and rapid motility (%), straight linear velocity (µm/s), straightness (%) reduced significantly (p < 0.05) when semen was diluted to 5 million sperm concentration. Among the various sperm function attributes, proportions of live spermatozoa, acrosome intact spermatozoa, hypo-osmotic swelling responsive spermatozoa and non-capacitated spermatozoa reduced (p < 0.05) in 5 million spermatozoa, and the proportions of moribund spermatozoa, dead spermatozoa, live acrosome reacted spermatozoa, dead acrosome intact spermatozoa, capacitated spermatozoa and spermatozoa with lipid peroxidation increased significantly (p < 0.05) when semen was diluted from 20 to 5 million. However, the over-dilution of semen did not affect slow motility, dead acrosome reacted spermatozoa, sperm protamine deficiency and spermatozoa with lipid peroxidation. In conclusion, the over dilution of semen affected sperm motion and functional attributes of frozen-thawed bull semen.

Cholesterol-loaded cyclodextrin attenuates dilution effect and improves quality of bovine low sperm insemination doses during cryopreservation.

In the present study, the effect of cholesterol-loaded cyclodextrin (CLC) on the quality of low sperm doses at post-thaw was evaluated. Twenty four ejaculates (6 from each bull) were collected and split into eight aliquots. First four aliquots were diluted up to 20-, 15-, 10- and 5-million sperm/0.25 ml, and remaining four were treated with CLC at the rate of 1 mg/120 million spermatozoa, followed by dilution up to 20-, 15-, 10- and 5-million sperm/0.25 ml. The diluted semen was equilibrated, cryopreserved and evaluated post-thaw. The averages of total motility, progressive motility, average path velocity, straight linear velocity, membrane intact spermatozoa and noncapacitated spermatozoa were higher (p < .05) in CLC-treated sperm doses compared to control ones. However, the moribund spermatozoa, capacitated spermatozoa and acrosome-reacted spermatozoa were reduced (p < .05) in CLC-treated spermatozoa compared to control. The curvilinear velocity and linearity did not differ (p > .05) between control and CLC-treated sperm doses. In conclusion, treatment of spermatozoa with CLC at the rate of 1 mg/120 million spermatozoon attenuates the dilution effect and improves the quality of bovine low sperm insemination doses during cryopreservation; hence it could be a favourable cryoprotectant for preserving bovine semen at higher dilutions.

Molecular characterization and protein structure prediction of heat shock transcriptional factors in goat (Capra hircus) and sheep (Ovis aries).

The heat shock factors are important as they are master regulator of heat shock response. There are only few mammalian HSFs which have been characterized, namely HSF-1, HSF-2, HSF-4 and HSF-5. The present study was aimed to clone and sequence characterize the partial open reading frames (ORFs) of HSF-2 and HSF-5 gene from cDNA isolated from testicular tissue of sheep (Macheri) and goat (Beetal). The partial ORFs of HSF-2 gene was observed to be 1627 bp in sheep and 1179 bp in goat and for HSF-5 it is 1137 bp in sheep and 1027 bp in goat. HSF-2 and HSF-5 encode a putative protein of 593 and 461 amino acid in goat and 568 and 553 amino acid in sheep, respectively. Phylogenetic analysis between the different orthologs suggested that these proteins are conserved from bovine to humans as well as in other mammals. Further, domain analyses using PredictNLS, MARCOIL and NetNES revealed that the members of HSF-2 protein orthologs contained all major domains, i.e., DNA-binding domain (DBD) and oligomerization domain (HR-A/B, and HR-C). The 3D structure of sheep and goat HSF-2 protein was predicted using SWISS-MODEL, which showed similar confirmation with the human HSF-2 protein sequence showing functional similarity between them.

Supplementing extender with anandamide enhances quality of low sperm doses during cryopreservation in bulls.

The present study explored the effect of anandamide supplementation in the extender on quality of low sperm doses during cryopreservation in Sahiwal bulls. Each fresh semen sample was split into eight aliquots (I, II, III, IV, V, VI, VII and VIII). The aliquots I, II, III and IV were taken as control and diluted to 20, 15, 10 and 5 million spermatozoa/0.25 ml respectively. The aliquots V, VI, VII and VIII were diluted with extender (supplemented with anandamide at 1 µM/ml of extender) to 20, 15, 10 and 5 million spermatozoa/0.25 ml respectively. This was followed by filling of diluted semen into French mini straws, equilibrated at 4°C of 4 hr and cryopreserved. The results revealed that the proportions of motile spermatozoa, live spermatozoa and live acrosome intact spermatozoa were significantly (p < .05) higher in all anandamide-treated sperm doses compared to control. The proportions of moribund spermatozoa, dead acrosome intact spermatozoa and capacitated spermatozoa were significantly (p < .05) reduced in all anandamide-treated sperm doses compared to control, with no difference in proportion of dead acrosome-reacted spermatozoa. In conclusion, anandamide supplementation in the extender increases the post-thaw quality of low sperm doses during cryopreservation in bulls.

Spermatozoa produced during winter are superior in terms of phenotypic characteristics and oviduct explants binding ability in the water buffalo (Bubalus bubalis).

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.

Sperm protein carbonylation.

The cryopreservation of sperm is a well established technique that plays an essential role in dissemination of elite germplasm of livestock. Despite having numerous advantages, the cryopreservation induces certain stresses on sperm including structural and functional damages leading to impaired sperm quality and fertility, which might be associated with production of reactive oxygen species (ROS). In addition, the ROS upon reacting with sperm lipids, DNA and proteins may lead to a cascade of sperm damages. The sperm membrane contains a rich amount of polyunsaturated fatty acids, which increases their susceptibility to oxidative stress induced damages, leading to formation of secondary products. These secondary products result in oxidation of sperm proteins via carbonylation. The carbonylation could lead to disturbances in specific proteins that are involved in capacitation. The present review deals with sperm protein carbonylation.

Salivary miR-16, miR-191 and miR-223: intuitive indicators of dominant ovarian follicles in buffaloes.

Estrus or sexual receptivity determination is utmost important for efficient breeding programs for female buffaloes. Prominent estrus behavioral symptoms are the result of several molecular and neuroendocrine events involving the ovary and the brain. Expression of estrus behavior is poor in buffaloes during the summer season. Hence, the discovery of biomarkers specific to the estrus stage or its related ovarian events, like the presence of dominant ovarian follicle, is helpful for developing an easy estrus determination method. MicroRNA are small non-coding RNA with a potential to be biomarkers. Therefore, the present study targeted to investigate the potential of estrogen responsive miRNAs (miR-24, miR-200c, miR-16, miR-191, miR-223 and miR-203) as estrus biomarkers in buffalo saliva, a non-invasive fluid representing animals' pathophysiology. There was a significant (P < 0.05) increase in the salivary presence of the miR-16, miR-191 and miR-223 at 6th and 18th-19th days than the 0 day (estrus), 10th day and the following consecutive estrus day. These observations may indicate an association between the representative lower presence of these miRNA in saliva and the presence of dominant ovarian follicles. To test this association, pathway analysis, target gene identification, functional annotation and protein-protein interaction networks (PPI) were performed for miR-16, miR-191 and miR-223 by different bioinformatics tools. Interestingly, the top pathways (fatty acid biosynthesis and oocyte meiosis), target genes (FGF, BDNF and IGF1) and PPI hub genes (KRAS, BCL2 and IGF1) of these miRNAs were found essential for ovarian follicular dominance. In conclusion, the miR-16, miR-191 and miR-223 may not be the perfect estrus stage-specific biomarkers. However, their lower presence in saliva at estrus and 9th-10th day of estrous cycles, when the ovary usually has a dominant follicle in buffaloes, may intuitively indicate the follicular dominance. Further studies are needed to prove this association in a large population.

Identification of potential protein biomarkers for early detection of pregnancy in cow urine using 2D DIGE and label free quantitation.

BACKGROUND: An early, reliable and noninvasive method of early pregnancy diagnosis is prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also help in to reduce the calving interval and rebreeding time which is beneficial for industries as well as farmers. The aim of this work is to identify potential biomarker for pregnancy detection at earlier stages (16-25 days). To achieve this goal we performed DIGE and LFQ for identification of protein which has significant differential expression during pregnancy. RESULTS: DIGE experiment revealed a total of eleven differentially expressed proteins out of which nine were up regulated having fold change ≥1.5 in all time points. The LFQ data analysis revealed 195 differentially expressed proteins (DEPs) out of 28 proteins were up-regulated and 40 down regulated having significant fold change ≥1.5 and ≤0.6 respectively. Bioinformatics analysis of DEPs showed that a majority of proteins were involved in regulation of leukocyte immunity, endopeptidase inhibitor activity, regulation of peptidase activity and polysaccharide binding. CONCLUSION: This is first report on differentially expressed protein during various time points of pregnancy in cow to our best knowledge. In our work, we identified few proteins such MBP, SERPIN, IGF which were differentially expressed and actively involved in various activities related to pregnancy such as embryo implantation, establishment and maintenance of pregnancy. Due to their involvement in these events, these can be considered as biomarker for pregnancy but further validation of is required.

Direct saliva transcript analysis as a novel non-invasive method for oestrus marker detection in buffaloes.

Salivary RNA-based biomarkers are not available for any physiological condition in farm animals. Hence, an objective of this study was to perform salivary transcript analysis in buffaloes. Saliva, after removal of the cells and particulate matter, was directly used for RT-PCR without RNA isolation. Direct saliva transcript analysis (DSTA) showed a suggestively significant higher expression of the Heat shock protein 70 (HSP70) and Toll-like receptor 4 (TLR4) at oestrus than the diestrous period in buffaloes by a non-parametric Mann-Whitney U test. Therefore, DSTA without RNA isolation is an easy method to identify salivary RNA markers for oestrus detection in buffaloes.

Mucin and salt combination simulate typical fern-like pattern of buffalo saliva smear at estrus.

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl.

Detection and quantification of TIMP1 and miR-141 through RT-LAMP and TT-LAMP in serum samples during estrous cycle in buffalo.

Estrus identification is a common problem in the reproductive management of farm animals. Hence, several studies have been conducted to explore biomarkers for estrus detection. One of our previous studies identified the abundance of RNA biomarkers such as TIMP1 and miR-141 in buffalo saliva during the estrus stage. However, the level of these RNA biomarkers in buffalo serum during estrous cycle is undetected. Therefore, the present study was designed to quantify TIMP1 and miR-141 in serum during buffalo estrous cycle. Blood samples were collected in different stages of estrous cycle from four healthy cyclic buffaloes. The quantification of TIMP1 and miR-141 was performed with direct serum using RT-LAMP and TT-LAMP technologies, respectively. The LAMP amplification was confirmed by agarose gel electrophoresis and the color change was quantified in comparison to a non-template control using ImageJ software. A decreased abundance of TIMP1 at the diestrus stage and a decreasing trend of miR-141 from proestrus to diestrus stages were observed, which was further reinforced by simulated random populations generated with R programming. Specifically, TIMP1 was found significantly (P < 0.0001) abundant at estrus and metestrus stages as compared to the diestrus stage, whereas miR-141 was significantly (P < 0.001) higher during the proestrus stage as compared to the other stages of estrous cycle. The ROC curve analysis showed miR-141 to be a better biomarker than TIMP1 as it distinguished the proestrus stage from diestrus with a sensitivity and specificity of 83 % and 98 %. This study also marked the first use of TT-LAMP technology for rapid miRNA detection in livestock.

miR-1246 is implicated as a possible candidate for endometrium remodelling facilitating implantation in buffalo (Bubalus bubalis).

BACKGROUND: The microRNAs (miRs) secreted by the trophectoderm (TE) cells have recently been implicated in the conceptus-endometrial cross talk during implantation and placentation. These miRs modulate various cellular processes during conception and throughout the pregnancy by regulating the gene expression in the foetal and maternal tissues. OBJECTIVES: This study was undertaken to elucidate the function of TE secreted miRNAs in the maternal-foetal cross-talk during implantation/placentation in buffalo. METHODS: The in vitro produced blastocysts were cultured on a cumulus feeder layer for 21 days. The relative expression profiles of a selected panel of miRs was generated using the spent media collected on Days 0, 7, 12, 16, and 21. A custom-designed mirVana™ miRNA mimic was used to transfect the endometrial epithelial cells (EECs) in order to determine the role of miRNA exhibiting highest expression on Days 21 and 21. RESULTS: The expression of miR-1246 (p < 0.001) and let-7b (p < 0.01) was found to be significantly higher on Day 21 of TE culture in comparison to the control (Day 0). This elevated expression indicated the involvement of these miRs in the maternal-foetal cross-talk. Interestingly, after the transfection of EECs with miRNA mimic for miR-1246 (a novel molecule vis-à-vis implantation), the expression of beta-catenin and mucin1 in these cells was found to be significantly (p < 0.05) downregulated vis-à-vis the control, that is, the IFN-τ primed EECs (before transfection). CONCLUSIONS: The TE secreted miR-1246 appeared to lower the expression of the endometrial receptivity genes (mucin1 and beta-catenin) which apparently assists the endometrium in preparing for placentation.

New dimensions on maternal and prepubertal nutritional disruption on bull fertility: A review.

Earlier, it was said that a bull is half of the herd because of its half contribution towards the genetic makeup in each subsequent generation. Nowadays, bulls are considered more than half of the herd because of the extensive use of frozen semen samples in artificial insemination. Bull's low fertility accounts for a major economic loss to livestock farmers. It is well known that fertility is a low-heritable trait governed by many factors such as genetics, epigenetics, climate, stress, and physical soundness. Apart from all these factors, the nutritional status of the bull also affects the semen quality. It has been seen that a bull given undernutrition at an early age is affected by androgen synthesis and semen quality. The nutrition given to the pregnant dam also affects the male progeny's postnatal semen quality. However, more studies are needed to elucidate the effect of periconception nutrition on the fertility of progeny as far as bulls are considered. This review focused on the effect of maternal undernutrition during the periconception period and undernutrition during the early growth phase of bull calves on the postnatal fertility of bulls.

The cryopreservation process induces alterations in proteins associated with bull sperm quality: The equilibration process could be a probable critical control point.

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

Comparative Proteome Profiling of Saliva Between Estrus and Non-Estrus Stages by Employing Label-Free Quantitation (LFQ) and Tandem Mass Tag (TMT)-LC-MS/MS Analysis: An Approach for Estrus Biomarker Identification in Bubalus bubalis.

Accurate determination of estrus is essentially required for efficient reproduction management of farm animals. Buffalo is a shy breeder and does not manifest overt signs of estrus that make estrus detection difficult resulting in a poor conception rate. Therefore, identifying estrus biomarkers in easily accessible biofluid such as saliva is of utmost interest. In the current study, we generated saliva proteome profiles during proestrus (PE), estrus (E), metestrus (ME), and diestrus (DE) stages of the buffalo estrous cycle using both label-free quantitation (LFQ) and labeled (TMT) quantitation and mass spectrometry analysis. A total of 520 proteins were identified as DEPs in LFQ; among these, 59 and four proteins were upregulated (FC ≥ 1.5) and downregulated (FC ≤ 0.5) during E vs. PE, ME, and DE comparisons, respectively. Similarly, TMT-LC-MS/MS analysis identified 369 DEPs; among these, 74 and 73 proteins were upregulated and downregulated during E vs. PE, ME, and DE stages, respectively. Functional annotations of GO terms showed enrichment of glycolysis, pyruvate metabolism, endopeptidase inhibitor activity, salivary secretion, innate immune response, calcium ion binding, oocyte meiosis, and estrogen signaling. Over-expression of SERPINB1, HSPA1A, VMO1, SDF4, LCN1, OBP, and ENO3 proteins during estrus was further confirmed by Western blotting. This is the first comprehensive report on differential proteome analysis of buffalo saliva between estrus and non-estrus stages. This study generated an important panel of candidate proteins that may be considered buffalo estrus biomarkers which can be applied in the development of a diagnostic kit for estrus detection in buffalo.