Microbial dynamics and climatic interactions in pig sheds: Insights into airborne microbes and particulate matter concentrations.

Emissions of airborne pollutants from livestock buildings affect indoor air quality, the health and well-being of farmers, animals and the environment. This study aimed to evaluate the microbial count within pig sheds and its relationship with meteorological variables (temperature, relative humidity and air velocity) and particulate matter (PM10 and PM2.5) and microbial diversity. Sampling was conducted both inside and outside of two pig sheds over three seasons (summer, rainy and winter), with regular monitoring at fortnightly intervals. Results showed that the bacterial and fungal counts ranged from 0.07 to 3.98 x 103 cfu/m3 inside the sheds and 0.01 to 1.82 x 103 cfu/m3 outside. Seasonal variations were observed, with higher concentrations of particulate matter detected during the winter season, followed by summer. Climatic variables such as temperature, air velocity and relative humidity demonstrated significant impacts on the abundance of Enterobacteriaceae and fungi, while air velocity specifically influenced the presence of mesophilic bacteria and staphylococci. Importantly, no significant disparities were found between microbial counts and particulate matter levels. Staphylococcaceae emerged as the predominant bacterial family, while Aspergillus and Cladosporium spp. were the dominant fungal species within the pig sheds. The average levels of airborne bacteria and fungi in pig sheds were found to be within the recommended range, which can be attributed to the loose housing design and lower animal population on the farms.

Effect of exogenous melatonin implant on post-thaw semen quality of buffalo bulls.

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa.

Additional intracervical prostaglandin reduces induction to calving time in buffaloes affected with incomplete cervical dilatation post-uterine detorsion.

Conventional induction protocol (CIP) of calving in buffaloes employs the intramuscular (IM) administration of dexamethasone (40 mg) and cloprostenol sodium (500 µg). If there is no progression in terms of cervical dilatation, then a second dose of cloprostenol sodium (500 µg) is administered intramuscularly. This protocol possesses certain demerits: (1) a wide range of response time intervals, and (2) increased risk of fetal membrane retention. Considering the cervix as a caudal continuation of the myometrium with its own contractile potential, and the limitations of CIP, we developed intracervical (IC) drug administration route in buffaloes. The proposed technique was evaluated for its use in a total of 22 cases of incomplete cervical dilatation in uterine torsion-affected buffaloes (IC-14 and IM-8). In addition to CIP, the IC group received an intracervical injection of cloprostenol sodium (500 µg) at the start of the experiment whereas the IM group received an extra intramuscular dose of cloprostenol sodium (500 µg) either after 24 h or when no progression in cervical dilatation is noticed. Surprisingly, the average response time during the experiment in the IC group was 5.8 h shorter (p < 0.000) than in the IM group (IC-5.7 ± 0.17 h vs. IM-11.9 ± 0.74 h). The duration from calving to fetal membrane expulsion (IC-12.8 ± 0.60 h vs. IM-17.5 ± 1.40 h; p < 0.002) and incidence of retention of fetal membrane were also less in the IC group (57.1% vs. 87.5%). The proposed intracervical drug administration potentiates cervical dilatation and can be regarded as a safe, effective, and feasible technique for attaining reliable results.

Local and systemic inflammatory response to the intrauterine infusion of enzymes during estrus in water buffaloes with subclinical endometritis.

Our objective was to determine the effects of intrauterine infusion of proteolytic enzymes in buffaloes with subclinical endometritis (SCE) at estrus on the resolution of endometrial inflammation and reproductive performance. Buffaloes at spontaneous estrus (E1) were screened for SCE by endometrial cytology to identify SCE (≥5% PMN, n = 22) and non-SCE (<5% PMNs, n = 14) animals. All buffaloes underwent uterine ultrasonographic examination, low volume uterine lavage (cytokines and acute phase proteins) and blood sampling (cytokines and acute-phase proteins) at E1. On the same day (E1), SCE buffaloes were randomly selected either for intrauterine infusion of proteolytic enzymes (ENY, n = 11) or saline (PC, n = 11). Buffaloes without SCE were kept as untreated control (NC; n = 14). All buffaloes were re-examined and re-sampled during subsequent estrus (E2), inseminated during the following estrus (E3), and assessed for fertility related outcomes. Proteolytic infusion resulted a reduction in uterine PMN (P < 0.01) in SCE buffaloes. The concentrations of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in uterus, and TNF-α and IL-10 in serum were higher (P < 0.01) at E1 in buffaloes with SCE (PC and ENY) compared to NC. After treatment, uterine IL-1ß and TNF-α (P = 0.02), and serum TNF-α and IL-10 were lower within the animals of ENY group (P < 0.01). Before treatment, buffaloes with SCE had higher concentrations (P < 0.01) of serum and uterine amyloid-A and haptoglobin, which decreased (P < 0.01) after treatment in the ENY group. None of the fertility outcomes differ between the treatment groups. In conclusion, intrauterine infusion of proteolytic enzymes reduced endometrial inflammation; however, did not improve reproductive outcomes.

A review on applications and toxicities of metallic nanoparticles in mammalian semen biology.

The advent of nanotechnology since the 1950s, when the well-known physicist Richard P. Feynman talked in his famous talk about "There's plenty of room at the bottom", has led to incredible contribution of nanotechnology in the fields of medical and veterinary therapeutics, diagnostics and other applications. Semen biology dealing with the study of spermatozoa and its related physiological and pathological aspects has not remained unscathed from the facets of nanotechnology. With each passing day investigators are revealing newer aspects of the nanoparticles, such as an antioxidants to relieve oxidative stress during semen cryopreservation, for the depletion of moribund spermatozoa from semen, gender selection of spermatozoa, bio-imaging of gametes, sperm mediated gene transfer, as well as for male fertility evaluation. As, the uses of various magnetic nanoparticles in the industry have gained acceleration, the evaluation of their effects, either beneficial or otherwise on the mammalian spermatozoa becomes obligatory. Many toxicological studies have also been conducted in respect to the harmful effects of different metallic nanoparticles related to their applicability, and industry borne adverse effects on the male germ cells in human beings and the animals. This review has been designed to focus on the beneficial as well as toxicological effects of various metallic nanoparticles on the mammalian spermatozoa and the future prospects related to their applicability in the semen biology.

Sodium dodecyl sulphate, N-octyl ß-D glucopyranoside and 4-methoxy phenyl ß-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls.

Sodium Dodecyl Sulphate (SDS), N-Octyl ß-D Glucopyranoside (NOG), 4-Methoxy Phenyl ß-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -40 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); ii) Moderate P25 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -50 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); and iii) Rapid P26 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -60 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo (Bubalus bubalis) bulls.

Graphene oxide (GO) greatly suppresses the growth and recrystallization by curving the hexagonal shape of ice crystals. Study was conducted to evaluate effect of GO as cryoprotectant in semen extender for augmenting sperm viability in dairy (cattle and buffalo) animals. In experiment one, semen was extended with TRIS Egg Yolk Glycerol (TEYG) extender supplemented with different concentrations of GO: 0.0125, 0.25, 0.5, 0.1 and 0.2 mg ml-1. Freezing of semen samples was conducted at 30 °C min-1 from temperature drop from 4 °C to -15 °C and -15 °C to - 60 °C followed by 50 °C min-1 from - 60 °C to -140 °C, and the semen straws were plunged in liquid nitrogen. Second experiment evaluated the performance of TEYG extender supplemented with combinations of GO (G05 as 0.05 and G10 as 0.1 mg ml-1) and glycerol (T48 as 4.8 and T64 as 6.4%) in four groups as G05T48, G05T64, G10T48 and G10T64. Freezing rates of 30 °C min-1[Protocol (PRT) I], 40 °C min-1 (PRT II) and 50 °C min-1 (PRT III) in the critical temperature fall zone of -15 °C to -60 °C were evaluated for semen extender supplemented with glycerol 6.4% and GO 0.05 mg ml-1 in the third experiment. Cattle (n = 3) and buffalo (n = 3) bulls were chosen for the study taking six ejaculates per bull per treatment. Post-thaw sperm motility, membrane integrity, viability and abnormalities were observed by means of CASA, Hypo-osmotic swelling test (HOST), Eosin-Nigrosin stain and Rose Bengal stain procedures, respectively. Post-thaw total motility (TM), progressive motility (PM), VCL, VSL, VAP, HOST response and viability increased significantly in extender with GO concentrations of 0.1 and 0.05 mg ml-1 as compared to control. Per cent abnormalities were significantly (p < .05) lower in group with GO 0.025 and 0.0125 mg ml-1 as compared to control. Results from the second experiment showed higher post-thaw TM, PM, VCL, VAP, VSL, HOST response, viability increased significantly (p < .05) in G05T64 and G05T48 as compared to G10T64. Sperm abnormalities did not vary among the groups as compared to control for cattle spermatozoa. In the third experiment post-thaw TM, PM, VCL, VSL, VAP, HOS response and sperm viability increased significantly (p < .05) in PRT III as compared to PRT I for buffalo and cattle spermatozoa. Sperm abnormalities were significantly (p < .05) lower in PRT II and PRT III as compared to PRT I for buffalo, whereas, lower in PRT II as compared to PRTI for cattle spermatozoa. GO as cryoprotectant when added to semen extender at the rate of 0.05 and 0.1 mg ml-1, resulted in better plasma membrane function and viability. Glycerol concentration below 6.4% in buffalo semen extender reduced post-thaw quality of sperm even when GO was added to the extender. Higher freezing rate of 50 °C min-1 in the critical temperature fall zone of -15 to -60 °C perform better than the freezing rate of 30 °C min-1. It is concluded that TEYG extender having glycerol 6.4% and GO 0.05 mg ml-1 improved post-thaw semen quality of cattle and buffalo.

Effects of intra-uterine infusion of proteolytic enzymes on selected cytokine concentrations, uterine inflammation, and fertility in postpartum water buffalo cows with subclinical endometritis.

The study was conducted to evaluate effects of intrauterine administration of proteolytic enzymes on endometrial inflammation and reproductive performance in postpartum water buffalo cows with subclinical endometritis (SCE). Cows (n = 38) with SCE (≥ 18 % PMN i.e.; polymorphonuclear cells) on day 21 postpartum (21 dpp), were allocated into treatment (TR; n = 19; intrauterine infusion of trypsin, chymotrypsin and papain in 20 ml normal saline on 21 dpp) and control (PC; n = 19; intrauterine administration 20 ml saline) groups. Cows without SCE (< 18 % PMN) were not treated and served as the negative control (NC; n = 30). Ultrasonography and sampling (endometrial cytology, uterine flushing, blood) were conducted on day 21 (before treatment) and 28 postpartum (28 dpp). The PMN % and uterine horn diameter were less on 28 dpp (compared with 21 dpp) in NC and TR group only. Cows with SCE had greater uterine concentrations of interleukin (IL)-1ß, IL-8 and tumor necrosis factor (TNF)-α; but lesser IL-10 than NC group on 21 and 28 dpp. There were greater serum IL-1ß and TNF-α concentrations on 28 dpp in cows with SCE than NC group. Uterine concentrations of IL-1ß were less, whereas IL-6 was greater following enzymatic treatment. Proteolytic enzyme treatment did not result in improvement in pregnancy rate compared with the PC group; however, days to conception were less in TR compared with the other two groups. In conclusion, results indicated a reduction in endometrial inflammation and days nonpregnant after proteolytic enzyme treatment in buffalo cows with SCE.

Evaluation of xanthosine treatment on gene expression of mammary glands in early lactating goats.

This study examined the hypothesis that xanthosine (XS) treatment would promote mammary-specific gene expression and stem cell transcripts and have a positive influence on milk yield of dairy goats. Seven primiparous Beetal goats were assigned to the study. Five days after kidding, one gland (either left or right) was infused with XS (TRT) twice daily for 3 d and the other gland with no XS infusion served as a control (CON). Mammary biopsies were collected at 10 d and RNA was isolated. Gene expression analysis of milk synthesis genes, mammary stem/progenitor cell markers, cell proliferation and differentiation markers were performed using real time quantitative PCR (RT-qPCR). Results showed that the transcripts of milk synthesis genes (BLG4, CSN2, LALBA, FABP3, CD36) and mammary stem/progenitor cell markers (ALDH1 and NR5A2) were increased in as a result of XS treatment. Average milk yield in TRT glands was increased marginally (approximately ~2% P = 0·05, paired t-test) per gland relative to CON gland until 7 wk. After 7 wk, milk yield of TRT and CON glands did not differ. Analysis of milk composition revealed that protein, lactose, fat and solids-not-fat percentages remained the same in TRT and CON glands. These results suggest that XS increases expression of milk synthesis genes, mammary stem/progenitor cells and has a small effect on milk yield.

Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein.

AIM: This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm. MATERIALS AND METHODS: A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 µM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity. RESULTS: There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29±4.23%) in Bcl-2 supplemented group (5 µM) as compared to control (51.6±5.77%). The proportion of HOST reactive sperms was also higher (63.1±6.73%) in Bcl-2 supplemented (5 µM) group as compared to control (50.7±6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 µM (73.42±5.79%), 10 µM (75.51±6.22%), and 15 µM (74.78±5.89%) as compared to control (60.23±4.45%). We found that Bcl-2 protein supplementation at 5 µM dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms). CONCLUSION: Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.