Reduced sperm number of murrah (Bubalus bubalis) bull semen in french mini-straw affects kinetic and functional competence after cryopreservation.

BACKGROUND: Extensive dilution of cattle semen with tris-based extender compromises certain sperm kinetic and functional traits following cryopreservation. OBJECTIVE: To study sperm functions of buffalo bulls under high dilution rates. MATERIALS AND METHODS: Twenty-four ejaculates were harvested twice a week from four buffalo bulls, and diluted to sperm concentrations of 80, 60, 40 and 20 million/mL. Diluted samples were filled in straws, equilibrated at refrigeration temperature for 4 h, and frozen in liquid nitrogen. Frozen sperm samples were thawed for evaluation of kinetic and functional attributes. RESULTS: Compared to 20 million/mL (million/mL) sperm sample, the total motility, progressive motility and rapid motility were reduced (P < 0.05) in 5 million/mL sample. The proportion of live sperm were significantly (P < 0.05) higher in 10, 15 and 20 million/mL samples than in 5 million/mL sample. The percentage of moribund sperm, dead sperm, and sperm with lipid per oxidation increased significantly (P < 0.05) in 5 million/mL sample. CONCLUSION: The reduction of sperm concentrations to < 10 million/mL affects post-thaw Buffalo sperm kinetic and functional attributes.. https://doi.org/10.54680/fr24110110712.

Comparative evidence support better antioxidant efficacy of mitochondrial-targeted (Mitoquinone) than cytosolic (Resveratrol) antioxidant in improving in-vitro sperm functions of cryopreserved buffalo (Bubalus bubalis) semen.

The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 µM, 25 µM, 50 µM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P < 0.05) in 200 nM MitoQ and 50 µM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 µM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P < 0.05) in MitoQ (200 nM) than RESV (50 µM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P < 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P < 0.05) and Bovine cervical mucus penetration was higher (P < 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P < 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P < 0.01) sperm with a higher reduction (P < 0.05) in membrane scrambling. MitoQ improved (P < 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P < 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.

Targeted transcript analysis revealed association of suboptimal expression of certain endometrial immunity-related genes with disparate uterine diseases in zebu cows.

The onset of uterine infection during postpartum period compromises uterine health, fertility, and productivity of dairy cattle. Endometrial innate immunity plays a key role in eliminating uterine infection and keeping the uterus healthy. Hence, the present study has been designed with the hypothesis that altered endometrial immune response around calving may compromise uterine health during postpartum period. Expression of interleukins (IL-1ß, IL-6, IL-8, IL-10, and TNF-α), prostaglandin synthase (PGFS, PGES), and antimicrobial peptides (beta-defensins (BDEF-4, BDEF-5), lingual antimicrobial peptide (LAP), and calcium-binding proteins (S100A8, S100A9, and S100A12) in endometrial tissues on the day of calving was studied using qRT-PCR, and circulating concentrations of prostaglandin E and F metabolites (PGEM and PGFM) during peripartum period (on days - 7, - 4, - 1 (before calving), 0 (on the day of calving), + 1, + 4, and + 7 (post calving)) of normal (healthy) cows (n = 11) that did not develop postpartum uterine infection and cows that developed puerperal metritis (n = 7) and clinical endometritis (n = 6) were studied. Endometrial expression of IL-1ß, TNF-α, BDEF-4, BDEF-5, S100A8, S100A12, and PGFS was higher (P < 0.05), and expression of IL-6, IL-8, IL-10, and PGES was lower (P < 0.05) in normal (healthy) cows than puerperal metritic and clinical endometritic cows. The PGFM concentration in serum was high (P < 0.05) on days 0, + 1, and + 4 of calving in puerperal metritic cows followed by normal and clinical endometritic cows. However, PGEM concentration in serum was high (P < 0.05) during peripartum period in uterine-infected (puerperal metritic and clinical endometritic) cows compared with normal cows. From the above findings, it is concluded that higher constitutive expression of IL-1ß, TNF-α, PGFS, BDEF-4, BDEF-5, S100A8, and S100A12 genes in the endometrium and lower concentration of PGEM during the period immediate to calving might be beneficial for uterine health of cows.

Improvement in sperm functional competence through modified low-dose packaging in French mini straws of bull semen.

To achieve the targeted artificial insemination coverage with the current rate of semen production, without affecting the conception rate, it needs to reduce the number of spermatozoa per insemination dose in India as per international practice. Therefore, this study was planned to perform different levels of semen dilution, compare in vitro post-thaw semen quality and develop a modified low-dose semen packaging method in French mini straw to minimise semen dilution effect. Sixteen ejaculates were collected from Karan Fries bulls (n = 4). The mean percentage post-thaw motility, viability, membrane integrity, acrosome integrity, lipid peroxidation and capacitation status were estimated as post-thaw sperm function assays in semen sample diluted to 20, 15, 10 and 5 million spermatozoa per 0.25 ml and filled in the French mini straw by conventional packaging. No significant (p > .05) difference in post-thaw sperm quality was observed between 15 and 20 million doses; however, below 15 million sperm quality get reduced. There was no significant difference in post-thaw semen quality traits between 20 million conventional packaging and 5 million spermatozoa/dose in modified packaging. In conclusions, the modified packaging is a very effective method for low-dose cryopreservation with acceptable post-thaw semen quality.

Global proteomic analysis of water buffalo (Bubalus bubalis) saliva at different stages of estrous cycle using high throughput mass spectrometry.

Accurate and efficient detection of estrus is one of the major constraints for exploitation of the production potential of buffalo owing to its poor manifestation of estrus signs, seasonal differences in expression and higher incidences of silent estrus (29%). The current study focused on identification of estrus specific candidate proteins in saliva of buffaloes. Estrus was detected based on behavioral signs in response to the teaser and changes in reproductive organs and confirmed by per-rectal examination, trans-rectal USG of reproductive organs, cervico-vaginal mucus characteristics and blood serum progesterone estimation. Day of onset of estrus was considered as day 0 and day -3, +3, +10 were considered as proestrus, metestrus and diestrus stage of the estrous cycle respectively. A total of 19 animals and their 38 estrous cycles (two from each) were included in this study. Saliva was collected from these animals during different stages of estrous cycle. Out of these, 08 animals were selected for global proteome analysis of saliva using in-solution digestion and nano-LC-MS/MS. A total of 275, 371, 304 and 565 proteins were identified with ≥2 peptides during proestrus, estrus, metestrus and diestrus stages of estrous cycle. Among the identified proteins 31, 62, 32 and 104 proteins were found specific to proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Few salivary proteins such as Cullin-associated NEDD8-dissociated protein 1, Heat shock 70 kDa protein 1A, 17-beta-hydroxysteroid dehydrogenase type 1, Inhibin beta A chain, testin were identified as estrus specific and are important for estrus physiology. Taken together, these estrus specific proteins could be considered as the candidate biomarker for detection and confirmation of estrus in buffalo after thorough validation.

Biochemical analysis of uterine fluid for identification of indicators for subclinical endometritis in the water buffalo (Bubalus bubalis).

Alterations in biochemical constituents of uterine fluid have been suggested for diagnosis of subclinical uterine infection in the bovine. This study was undertaken to investigate whether uterine fluid biomolecules could act as tool for diagnosis of subclinical endometritis in the buffalo. Uterine fluid samples from normal (n = 22) and subclinical endometritis (n = 18; diagnosed based on uterine cytology)-affected buffaloes were subjected to biochemical analysis. Among the different biochemical constituents estimated, urea, urea N, cholesterol, total bilirubin and alkaline phosphatase (ALP) concentrations were significantly (p < 0.05) higher in uterine fluid obtained from subclinical endometritis-affected buffaloes. The extent of difference between normal and subclinical endometritis-affected buffaloes was highest in ALP (69%) followed by cholesterol (55%), bilirubin (48%), urea (30%) and urea N (30%) concentrations. Receiver operating characteristic (ROC) analysis indicated that the likelihood ratio (LR) was 3.63 for urea, indicating that buffaloes having less than the threshold concentration (47.5 mg/dl) of urea in their uterine fluid were at 3.6 times more risk to be affected with SE. The LRs for urea N, cholesterol, ALP and bilirubin were 2.33, 2.54, 2.12 and 1.65, respectively. It was concluded that ALP, urea, urea N and cholesterol concentrations in uterine fluid may serve as an aid for diagnosing subclinical endometritis in the buffalo.

Age-related changes in transcriptional abundance and circulating levels of anti-Mullerian hormone and Sertoli cell count in crossbred and Zebu bovine males.

Age-related changes in peripheral anti-Mullerian hormone (AMH) concentrations and transcriptional abundance of AMH gene in testicular tissue were studied in crossbred (Holstein Friesian × Tharparkar) and Zebu (Tharparkar) males. In both the breeds, basal AMH concentrations were estimated using ELISA method in blood plasma obtained from six males each at 1, 6, 12, 18, and 24 months age. After blood collection at respective ages, all the males were castrated and expression and immunolocalization of AMH was performed in the testicular tissue. The concentration of AMH in blood plasma was found to be highest at 1 month of age in both crossbred and Zebu males, which subsequently decreased with advancing age. Significantly (P < 0.05) lower concentration of AMH was observed in crossbred as compared with Zebu males at 24 months of age. In line with peripheral AMH concentrations, the expression of AMH gene was also higher (P < 0.05) at 1 month of age, which thereafter declined significantly with advancement of age in crossbred males. Furthermore, the expression of AMH gene differed significantly between Zebu and crossbred males at all the age groups studied. Immunolocalization of AMH in testicular tissue also revealed a stronger expression at 1 month age, which gradually decreased till 24 months of age. The true Sertoli cell count was significantly higher in Zebu compared with crossbred males at all age groups studied except at 6 months age. The relationship between Sertoli cell count and circulating AMH concentrations was negative and significant (r = -0.81; P = 0.004). In conclusion, expression of AMH gene in testicular tissue and peripheral blood concentrations of AMH were higher in young compared with adults in both crossbred and Zebu males; however, the transcriptional abundance and circulating levels of AMH were higher in Zebu compared with crossbred males.

Effect of Dilution on Cryosurvival of Low Sperm Doses: A Review.

　　BACKGROUND: Sperm concentration needed for artificial insemination (AI) to obtain reasonable fertility, taking into consideration genetic value of bull and numerous others components is one of the essential constituents for successful breeding program. AI-doses containing low sperm numbers are increasingly widespread to optimize the benefit of elite bulls, as well as to accommodate an eventual wider application of sex-sorted semen. Viability is reduced when higher dilutions of semen are carried out for production of low sperm doses. The reduction in viability may be due to dilution effect as well as due to dilution of numerous essential constituents in seminal plasma at higher dilutions. Fertility level of bull may play a pivotal role in determining the cryosurvival of low sperm doses and viability of low sperm doses may vary among bulls. This paper attempts to highlight studies dealing with the effect of dilution on cryosurvival of low sperm doses.

Transcriptional abundance of antioxidant enzymes in endometrium and their circulating levels in Zebu cows with and without uterine infection.

Oxidative stress during peripartum period may compromise the uterine immunity. In the present study, we assessed the oxidative stress and antioxidant status during peripartum period and studied their relationship with postpartum uterine infection in dairy cows. Peripheral blood concentrations of total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) were determined (day -21, -7, on the day of calving and day +7, +21, +35) in normal (n=11), puerperal metritic (n=7) and clinical endometritic (n=6) cows. Endometrial biopsy was performed on the day of calving and expression of CAT, GPx4 and SOD2 genes was studied using qRT-PCR. Puerperal metritic cows had significantly (P<0.05) lower TAC (on day -7, day 0, day +7, +21 & +35), higher MDA (on day -21, -7 & on the day of calving) and NO (on day 0, +7 & day +35) concentrations compared to normal cows. Similarly, clinical endometritic cows had significantly (P<0.05) lower TAC (on day -7, 0, +7 & +21), higher MDA (on day -21, -7, +7 and +35) and NO (on day +7, +21 & +35) concentrations compared to normal cows. The expression of CAT and GPx4 genes was lower (P<0.05) and SOD2 gene was higher (P<0.05) in endometrial tissue of cows that developed uterine infection compared to normal cows. The relationship of peripheral levels of MDA and NO with antioxidant enzymes expression in endometrial tissue was found significant. Receiver operator characteristic analysis revealed that the concentrations of TAC on day -7 to day +35, MDA on day -21 to day +7 and NO on the day of calving to day +35 were highly correlated to the development of postpartum uterine infection in cows. It may be inferred that the low serum TAC level and high level of lipid peroxidation and NO during peripartum period influenced the endometrial expression of anitioxidative genes that compromised the uterine health during postpartum period.

Efficiency of uterine fluid cytology in the diagnosis of subclinical endometritis in the water buffalo (Bubalus bubalis).

This study compared endometrial cytology vis-a-vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)-affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE-affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.

Modulation of post-partum reproductive performance in dairy cows through supplementation of long- or short-chain fatty acids during transition period.

Thirty-six cross-bred cows were used to study the effect of long-chain (flaxseed) or short-chain (butyric acid) fatty acid supplementation on metabolic status, ovarian function and reproduction performance during transition period. Control cows received a routine feed of transition diet, while the cows in two treatment groups were supplemented with either 750-g crushed flaxseed or 250 g butyric acid per cow per day. Ovarian activity was monitored by transrectal ultrasonography on 10th, 20th and 30th days post-partum. Blood samples were collected by jugular venipuncture into heparinized polystyrene tubes; plasma was prepared and stored under -20 °C until analysis. Results indicated that cows in flaxseed group were in positive energy balance as indicated by lower NEFA and Beta hydroxy Butyrate and higher glucose concentrations. Uterine involution was completed well within 30 days post-partum in all the cows in flaxseed fed group compared to 76.9% in butyric acid supplemented and 61.5% in control groups. The size of dominant follicle and corpus luteum was significantly higher (p < 0.05) for flaxseed group compared to control group, which in turn resulted in higher concentrations of plasma progesterone. Cows fed on diets supplemented with flaxseed exhibited post-partum heat earlier and bred sooner (p < 0.05) than control cows. It has been noticed that supplementation of flaxseed and butyric acid enhanced involution of uterus, early resumption of cyclicity and thereby early breeding. However, in view of the encouraging results obtained for flaxseed supplemented group, its organic nature and easier availability at farmer's gate, we concluded that flaxseed can be safely included in transition diet to modulate reproductive performance of dairy cattle.

Alteration in peripheral blood concentration of certain pro-inflammatory cytokines in cows developing retention of fetal membranes.

Retention of fetal membranes (RFM) adversely affects the production and reproduction potential of the affected cows leading to huge economic loss. Physiological separation of fetal membranes is reported to be an inflammatory process. The present study compared the concentrations of certain pro inflammatory cytokines [Interleukin 1ß (IL-1), Interleukin 6 (IL-6), Interleukin 8 (IL-8) and Tumor necrosis factor α (TNF-α) between the cows that developed RFM (n=10) and the cows that expelled fetal membranes normally (n=10) to find out if they could serve as a predictive tool for RFM. Blood samples were collected from the cows from 30 days before expected parturition through day -21, day -14, day -7, day -5, day -3, day -1, on the day of parturition (day 0), day 1 postpartum and the pro-inflammatory cytokines were estimated in blood plasma by ELISA method. The IL-1ß concentration was significantly lower (P<0.05) in cows that developed RFM compared to those that expelled fetal membranes normally from 3 days before calving till the day of calving. The plasma concentrations of IL-6 and IL-8 were also lower (P<0.05) in cows that developed RFM than those calved normally. On the day of calving, significantly (P<0.05) lower concentrations of TNF-α was observed in cows that developed RFM compared to those expelled fetal membranes normally. It may be inferred that the concentrations of IL-1, IL-6, IL-8 and TNF-α around parturition were altered in cows developing RFM compared to those expelled fetal membranes normally.

Expression of short chain fatty acid receptors and pro-inflammatory cytokines in utero-placental tissues is altered in cows developing retention of fetal membranes.

INTRODUCTION: Retention of fetal membranes (RFM) is an economically important condition in dairy cattle disturbing the health and production in affected animals. In spite of extensive research, pathogenesis for RFM and identification of a predictive marker for the condition still remains elusive. Since expulsion of fetal membranes in an inflammatory process, studying the expression of inflammatory molecules is expected to give an idea about the pathogenesis of RFM. METHODS: Expression of pro-inflammatory cytokines, short chain fatty acid (SCFA) receptors and adhesion molecules was studied using qRT-PCR in the utero-placental tissues obtained from the cows that expelled fetal membranes normally (n = 12) and from the cows that developed RFM (n = 12). Concentrations of SCFAs were estimated in the utero-placental tissues using Gas Chromatography. RESULTS: The concentration of butyrate in the utero-placental tissue extract was 3-4 times lower, and GPR41 and GPR43 mRNA expression was 7.7-10.0 and 3.4-4.4 folds lower (p < 0.05) in cows that developed RFM compared to those that expelled the fetal membranes normally. The expression of IL-1, IL-6, IL-8, TNF-α, ICAM and PECAM was higher (p < 0.05) in the cows that normally expelled the fetal membranes compared to those that developed RFM. DISCUSSION: Since a clear association of butyrate levels, expression of pro-inflammatory cytokines, adhesion molecules and SCFAs receptors was observed with expulsion of fetal membranes, modulating the levels of these molecules prior to parturition might help in reducing the incidence of RFM in cows. CONCLUSION: Inflammatory response is essential for normal expulsion of fetal membranes and that inadequate or altered expression of pro-inflammatory cytokines, adhesion molecules and SCFA receptors is associated with RFM in cows.