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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Open Access Study on the effects of prostaglandin therapy in combination with antiprogestin and antiprolactin treatments for therapeutic management of pyometra in She Dogs(LUVAS Hisar, 2023-04) Mohit Kumar; Harpreet SinghThe present research work entitled “Study on the effects of prostaglandin therapy in combination with antiprogestin and antiprolactin treatments for therapeutic management of pyometra in She Dogs” was carried out in the veterinary clinical complex, LUVAS, Hisar. Diagnosis of pyometra was done by correlating the history and clinical signs with the findings of abdominal palpation and ultrasonography. A total of 28 she dogs diagnosed for pyometra were randomly assigned to four treatment protocols with 7 animals in each group. In Group-I, all the animals were administered with antibiotic Enrofloxacin @ 10 mg/kg body wt., intramuscularly, OD for 5 days. In Group-II, all the animals were administered with cloprostenol (@2.5 µg/kg body wt., S/C on 1st, 3rd and 5th day) along with Enrofloxacin @ 10 mg/kg body wt., intramuscularly, OD for 5 days. In Group-III, all the animals were administered with cabergoline (@ 5µg/kg body wt., orally OD for five days) and cloprostenol (@2.5 µg/kg body wt., S/C on 1st, 3rd and 5th day), alongwith Enrofloxacin @ 10 mg/kg body wt., intramuscularly, OD for 5 days. In Group-IV, all the animals were administered with Mifepristone @ 2.5 mg/kg body wt., orally BID for 5 days, and cloprsotenol (@2.5 µg/kg body wt., S/C on 1st, 3rd and 5 th day) alongwith Enrofloxacin @ 10 mg/kg body wt., intramuscularly, OD for 5 days. Ultrasonographic and hematobiochemical evaluations were performed on day 0 and day 7 to assess efficacy of the treatment protocols. The most common clinical signs recorded in the she dogs were vaginal discharge (92.86%), followed by anorexia (75%), vomition (53.57%), polyuria (39.29%) and polydipsia (35.71%). Ultrasonography revealed an enlarged uteri with convoluted, tubular horns filled with anechoic to hypoechoic fluid and the mean size of pus filled uterine cavities decreased significantly (p<0.05) at the end of treatment (day 7) in she dogs of the groups-II, -III and-IV. The total leucocyte and neutrophil counts were increased in the she dogs affected with pyometra which showed significant (p<0.01) decline at end of treatment in she dogs of the groups-I, -III and -IV. Vaginal discharge samples were mostly sensitive to Gentamicin (57.14%) and Enrofloxacin (53.57%) antibiotics. Pre-treatment biochemical profiles do not show any distinctive changes; except mild elevation in blood urea nitrogen (BUN) and alkaline phosphatase. Serum aspartate transaminase concentations decreased significantly (p<0.05) in she dogs of the group-I and -III after the treatment. Seum ALT and Serum alkaline phosphatase profiles decreased significantly (p<0.05) after the treatment in group-III. After completion of treatment, the highest recovery rate (71.42%) from pyometra was observed in she dogs of the group-III as well as group-IV, whereas the recovery rate in group-I and group-II was lower, 42.85% and 57.14%, respectively. Two out of four recovered she dogs in the group-II came to estrus. In conclusion, the combination of prostaglandin analogues (Cloprostenol), antibiotic therapy alongwith anti-progestin (Mifepristone) or anti-prolactin (Cabergoline) hormones is an effective approach for medicinal treatment of canine pyometra.ThesisItem Open Access Influence of antibiotic substitutes on bacterial load and semen quality of buffalo bulls(LUVAS Hisar, 2023-05) Usha yadav; Dutt,RaviThe aim of the present study was to evaluate the bacterial load and semen quality of buffalo bull semen in fresh and cryopreserved semen samples by using extenders supplemented with antibiotics, without antibiotics and substitutes of antibiotics. For this study, ejaculates were collected from sixteen Murrah bulls and two experiments were performed. The second experiment was further divided into two independent experiments (1) and (2) respectively. For the estimation of microbial load 80 ejaculates were collected through AV method for experiment 1 and semen quality was assessed in 16 samples, whereas 5 and 16 ejaculates respectively for experiment (1) and (2) for estimation of microbial load and semen quality. For experiment 1, each ejaculate was divided into two aliquots: 1) control (NAB), 2) extender supplemented with antibiotics (AB). For experiment (1) of second experiment, semen samples were diluted in extenders supplemented with KA (0.02, 0.04, 0.06, 0.08 and 0.1g/L), AB and control (NAB), for experiment (2), extenders supplemented with epsilon-polylysine (0.64 and 1.28g/L), AB and control (NAB) and cryopreserved. After post-thaw, the following parameters were studied; thermal resistance of sperm by incubation test, sperm motility and kinetics by (CASA), acrosome integrity, mitochondrial membrane potential (MMP) and mitochondrial superoxide levels through flow cytometry. The results of the present study showing a non-significant difference in bacterial load (CFU/mL) and a non-significant effect on sperm motility and kinetic parameters, plasma membrane integrity, HMMP and increased superoxide levels in AB and NAB groups. There was no significant difference in endotoxin levels estimated by kit in both the groups. The results of different treatment groups of Kojic Acid in comparision to AB and NAB showed non-significant difference in bacterial load and sperm quality parameters. The results of different treatment groups of epsilon-polylysin showed non-significant decrease in bacterial load whereas quality parameters were improved significantly. In conclusion, the microbial load was controlled in semen extenders without the addition of antibiotics and sperm quality parameters parameters were equivalent in group AB and NAB. Addition of Kojic acid in semen extender did not show any antibacterial effect whereas epsilon-polylysin showed antibacterial effect and improved semen quality.ThesisItem Restricted Effect of lipopolysaccharide on in vitro development of buffalo oocytes and embryo development(2023-02-13) Sujata; Ravi DuttThe study investigated the effect of lipopolysaccharide (LPS) on in vitro maturation (IVM) and in in vitro culture (IVC) media on buffalo oocytes and in vitro fertilized (IVF) blastocysts. Effect of LPS on maturation rate, mitochondrial membrane potential (MMP), ROS production, apoptosis, gene expression (inflammatory, apoptosis, antioxidant), embryonic development and total cell number (inner cell mass and trophectoderm) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The different concentrations of LPS were supplemented in IVM (0, 5, 10 and 20μg/mL) and IVC (0 and 5μg/mL) media. Supplementation of LPS in IVM media reduced the cumulus expansion, germinal vesicle breakdown, MMP and the expression of antioxidant genes (SOD2 & GPX1), whereas ROS production, expression of inflammatory genes (TLR4, CD14, RPS27A) and apoptotic gene (Caspase 3) increased significantly (p<0.001) in a dose dependent manner resulting in decreased oocytes maturation rate. LPS supplementation in IVC media reduced the development competency and blastocyst quality of IVF embryos by decreasing the MMP, total cell number, expression of antioxidant and pluripotent gene and increasing the ROS production, inflammatory and apoptotic gene. The results of the present study suggested that LPS exposure on IVM or IVC media @ 5μg/ml impaired the oocytes maturation and also reduced developmental competence of blastocysts with compromising their qualityThesisItem Restricted Influence of antibiotic substitutes on bacterial load and semen quality of buffalo bulls(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-05) yadav, Usha; Dutt, RaviThe aim of the present study was to evaluate the bacterial load and semen quality of buffalo bull semen in fresh and cryopreserved semen samples by using extenders supplemented with antibiotics, without antibiotics and substitutes of antibiotics. For this study, ejaculates were collected from sixteen Murrah bulls and two experiments were performed. The second experiment was further divided into two independent experiments (1) and (2) respectively. For the estimation of microbial load 80 ejaculates were collected through AV method for experiment 1 and semen quality was assessed in 16 samples, whereas 5 and 16 ejaculates respectively for experiment (1) and (2) for estimation of microbial load and semen quality. For experiment 1, each ejaculate was divided into two aliquots: 1) control (NAB), 2) extender supplemented with antibiotics (AB). For experiment (1) of second experiment, semen samples were diluted in extenders supplemented with KA (0.02, 0.04, 0.06, 0.08 and 0.1g/L), AB and control (NAB), for experiment (2), extenders supplemented with epsilon polylysine (0.64 and 1.28g/L), AB and control (NAB) and cryopreserved. After post-thaw, the following parameters were studied; thermal resistance of sperm by incubation test, sperm motility and kinetics by (CASA), acrosome integrity, mitochondrial membrane potential (MMP) and mitochondrial superoxide levels through flow cytometry. The results of the present study showing a non-significant difference in bacterial load (CFU/mL) and a non-significant effect on sperm motility and kinetic parameters, plasma membrane integrity, HMMP and increased superoxide levels in AB and NAB groups. There was no significant difference in endotoxin levels estimated by kit in both the groups. The results of different treatment groups of Kojic Acid in comparision to AB and NAB showed non significant difference in bacterial load and sperm quality parameters. The results of different treatment groups of epsilon-polylysin showed non-significant decrease in bacterial load whereas quality parameters were improved significantly. In conclusion, the microbial load was controlled in semen extenders without the addition of antibiotics and sperm quality parameters parameters were equivalent in group AB and NAB. Addition of Kojic acid in semen extender did not show any antibacterial effect whereas epsilon-polylysin showed antibacterial effect and improved semen quality.ThesisItem Restricted Effect of lipopolysaccharide on in vitro development of buffalo oocytes and embryo developmen(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Sujata; Dutt, RaviThe study investigated the effect of lipopolysaccharide (LPS) on in vitro maturation (IVM) and in in vitro culture (IVC) media on buffalo oocytes and in vitro fertilized (IVF) blastocysts. Effect of LPS on maturation rate, mitochondrial membrane potential (MMP), ROS production, apoptosis, gene expression (inflammatory, apoptosis, antioxidant), embryonic development and total cell number (inner cell mass and trophectoderm) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The different concentrations of LPS were supplemented in IVM (0, 5, 10 and 20μg/mL) and IVC (0 and 5μg/mL) media. Supplementation of LPS in IVM media reduced the cumulus expansion, germinal vesicle breakdown, MMP and the expression of antioxidant genes (SOD2 & GPX1), whereas ROS production, expression of inflammatory genes (TLR4, CD14, RPS27A) and apoptotic gene (Caspase 3) increased significantly (p<0.001) in a dose dependent manner resulting in decreased oocytes maturation rate. LPS supplementation in IVC media reduced the development competency and blastocyst quality of IVF embryos by decreasing the MMP, total cell number, expression of antioxidant and pluripotent gene and increasing the ROS production, inflammatory and apoptotic gene. The results of the present study suggested that LPS exposure on IVM or IVC media @ 5µg/ml impaired the oocytes maturation and also reduced developmental competence of blastocysts with compromising their qualityThesisItem Restricted Comparative study on efficacy of Prostaglandin F2α, Estradiol benzoate and Buserelin acetate based estrus synchronization protocols on reproductive performance in cyclic buffaloes(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-05) YADAV, VINAY; Pandey, A.KThe present study was done with the hypothesis that use of short term protocols with variable combinations of PGF2α, EB and GnRH could modify the exiting follicular wave with resultant improved pregnancy rate in the normal cyclic animals. The study was planned with four different protocols viz. Group-I (n=40): d0: PGF2α; Group-II (n=54): d0: PGF2α, d1: EB; Group-III (n=53): d0: PGF2α, d2: GnRH; and Group-IV (n=66): d0: PGF2α, d3: GnRH with the objectives to study pregnancy rate, endocrine profile before and after AI and ovarian activity in response to these protocols in normal cyclic buffaloes in diestrus phase. The FSCR (%) varied non-significantly (p>0.05) among all groups but Group-III had highest FSCR (43.4%; 23/53) followed by 33.33% (18/54) in Group-II, 31.82% (21/66) in Group-IV and lowest 27.5% (11/40) in Group-I. The animals exhibited intense estrus response in Group-I (7.50%; 3/40) were significantly (p0.05) to Group-I, III and Group-IV. The correlation coefficient of CL area on day 5 and 12 post-AI was 0.39 (p<0.05) and 0.44 (p<0.05) with POF diameter. It was concluded that administration of short term estrus synchronization protocols resulted in 100% estrus induction, with variable combination of PGF2α and GnRH also have almost similar pregnancy rates in buffaloes as compared to longer FTAI protocols developed earlier. The intensity of estrus response, greater concentration of estradiol and lesser concentration of progesterone on day of AI had positive association with increased pregnancy rate.ThesisItem Open Access Studies on minimization of cryocapacitation of buffalo (Bubalus bubalis) sperm during cryopreservation(LUVAS, 2019) JASMER; CHANDOLIA, R.K.Keeping fact in mind that cryocapacitation is detrimental to sperm, the present study was designed to minimize the cryocapacitation in buffalo bull sperm by using low density glycoprotein (LDL) in place of yolk, mifepristone (RU 486), NNC 55-0396 (NNC), H-89 and EC Oxyrase in order to protect sperm membrane, antagonising progesterone of egg yolk, minimize Ca2+ influx, inhibit signalling molecule protein kinase A (PKA) and oxidative stress by digesting O2 during cryopreservation, respectively. The LDL (10, 12 and 14%) was used to replace 20% of egg yolk (EY) in the semen extender. The semen ejaculates having mass motility (MM) ≥ 3.0 and Individual motility ≥ 75% were cryopreserved in EY and LDL extenders. Further, LDL 12 % was compared with egg yolk to cryopreserve the poor and marginal quality ejaculates (MM ≤2.5; TM ≤ 65%). We also measured the progesterone and Ca2+ in LDL and EY. The ejaculate was divided into four equal aliquots and extended using egg yolk- based extender fortified with different concentration of RU 486 (0, 5, 10 and 20 μM), NNC (0, 2, 5 and 10 μM), H-89 (0, 2, 10, 30 μM) and Oxyrase (0, 0.3, 0.6, 0.9, 1.2 u/mL) separately and cryopreserved. The semen samples were analysed for sperm kinetics and motility, incubation test, integrity of sperm membrane (HOST), sperm cholesterol, expression of CatSper-2 proteins (LDL and mifepristone), Ca2+ influx in sperm, expression of tyrosine phosphorylated proteins (TPP), cryocapacitation of sperm (CTC assay), lipid peroxidation (MDA concentration), and antioxidative capacities of LDL and mifepristone through FRAP assay. Differential expression of CatSper-1, CatSper-2, e-NOS, n-Nos and c-Myc was also assessed in sperm cryopreserved in LDL and EY extender. Further mifepristone, NNC and H-89 treated samples were analysed for in vitro capacitation and zona binding ability. Moreover, mifepristone treated sperm were also analysed for in vitro fertilizing potential. We found that progesterone and Ca2+ were extremely low in LDL as compared to EY (P<0.05). Sperm motility and kinetic parameters were better in LDL as compared to EY. The RU 486, NNC and H-89 and NNC did not affect the sperm motility (P>0.05). But, LDL and Oxyrase prolonged the sperm longevity (P<0.05). The LDL and RU 486 protected the sperm membrane integrity (P<0.05). The LDL, RU 486, NNC, H-89 and Oxyrase prevented the efflux of cholesterol, influx of Ca2+ (P<0.05). Lipid peroxidation of sperm membrane was lower as indicated by lower MDA concentration in sperm cryopreserved in LDL extender and in sperm treated RU 486, NNC and Oxyrase as compared to control (P<0.05). The expression of CatSper-2 proteins was higher in sperm cryopreserved in LDL extender and sperm treated with mifepristone as compared to EY (P<0.05). The expression of TPP was lowered in sperm cryopreserved in LDL as compared to EY (P<0.05). Also, expression of TPP was lowered in sperm treated with RU 486, NNC, H-89 and Oxyrase (P<0.05). The RU 486, NNC and H-89 treated sperm were shown normal in vitro capacitation and attached tightly to zona pellucida (P<0.05) as compared to control. Also, RU 486 treated sperm had better in vitro fertilization rate as compared to control (P<0.05). LDL and RU 486 fortified extender showed more antioxidant power based on DDPH and FRAP assays. The expression of CatSper-1, CatSper-2, n-NOS and c-Myc were upregulated where as that of e-NOS was downregulated in sperm cryopreserved in LDL extender as compared to EY. The LDL 12 % extender was more efficient in successful cryopreservation of poor and marginal ejaculates as compared to EY (P<0.05). Taken all together, LDL, RU 486, NNC, H-89 and Oxyrase in the present study found to beneficial in preventing the cryocapacitation in buffalo bull sperm.ThesisItem Open Access Microbiological, Cytological and Ultrasonographic studies in infertile mares and their therapeutic management(LUVAS, 2019) Nafis Ibni Assad; . N S BugaliaInfertility in broodmares due to uterine infection and associated endometritis occurs following breeding and foaling, and cripples annual foal production and accounts for heavy economic loss in the equine industry. Both diagnosis and therapeutic management as early as possible are warranted to maintain optimum fertility. Present study was therefore undertaken with the aim to evaluate the diagnostic utility of uterine cytology, uterine culture and ultrasonography in detection of uterine infection in infertile mares and to recommend a combination of diagnostic techniques to be employed as a routine procedure in the management of equine uterine infections and associated endometritis. The therapeutic efficacy of different treatment regimens in management of uterine infection in infertile mares was also evaluated. Investigation was conducted on 27 broodmares of equine breeding stud (RVC)-Hisar. Mares were divided into four groups (Group I, II and III, with 7 infertile mares in each group; Group IV with 6 fertile mares). All the mares were subjected to pre-treatment assessment of reproductive status by uterine ultrasonography, uterine cytology and uterine culture, followed by therapeutic management of uterine infections and associated endometritis with three different therapeutic regimens for three different groups. Transrectal ultrasound was done using a linear array probe (5MHz and 7 MHz). Sampling for uterine cytology and uterine cultures was done using low-volume uterine flush technique. Group I mares were treated with saline uterine lavage + PGF2α + intrauterine antibiotic infusion, Group II mares received saline uterine lavage + Cloprostenol + intrauterine antibiotic infusion, Group III mares were treated with saline uterine lavage + Oxytocin + intrauterine antibiotic infusion. Group IV mares received no treatment. The antibiotics used were based on the in vitro antibiotic sensitivity test. Further, therapeutic response was evaluated by post-treatment ultrasound conducted between days 7-10 after treatment and post-treatment conception was recorded following 1st, 2nd and 3rd inseminations. The sensitivity, specificity, positive predictive value and negative predictive value of ultrasound, uterine cytology & uterine culture were recorded to be 86.66%, 50.00%, 68.42% & 75.00%; 71.42%, 69.23%,71.4% & 69.23% and 100%,100%, 100% & 100% respectively. The results of all the techniques were highly and significantly correlated. Uterine cultural examination of the flush sample was the most reliable method for detection of uterine infection in infertile mares. Uterine culture and uterine cytology was found to be the best combination for routine use in the diagnosis of infections. However, uterine ultrasonography may be used for evaluation of quantity of uterine fluid as well as assessment of endometrial oedema/uterine wall thickening in prediction of fertility. Therapeutic regimens involving Cloprostenol and Oxytocin in addition to uterine lavage + intrauterine antibiotic infusion were found to be first and second preference treatment protocols, respectively, in management of uterine infections in infertile mares.ThesisItem Open Access Three dimensional (3D) ultrasonographic and color Doppler studies of prenatal fetal development in ewe and postnatal testicular development in ram.(LUVAS, 2019) Sandeep Kumar; CHANDOLIA, R.K.The present study was conducted on 2 to 2.5 year age six Harnali ewes and Six Harnali prepubertal rams maintained at sheep breeding farm, Deptt. AGB, LUVAS, Hisar. Rams were introduced to them and matings were monitored and day of mating considered as day 0. Sequential ultrasonographic scanning was conducted twice a week from day 20 to day 60, then weekly till the end of experiment in ewe to study the fetal development. To study testicular development in lamb ram, scanning was done bimonthly from 2 weeks to 4 month; then weekly from 4 months to 6 months. The ultrasonographic images were obtained using Toshiba Nemio-XG 3D ultrasound machine with intra-operative probe with 7.5 MHz frequency, convex 2D (5MHz) and 3D volumetric probe was used in both studies. The embryo and amniotic vesicle were first observed on day 23 and 29, respectively. Complete organization of the embryo into head, body and limb buds was detected by day 38. Ossification elements was observed in skull, mandible, limbs, vertebral column and nasal bone on day 41, 44, 44, 50 and 56, respectively. The fetal crown-rump length (CRL), BPD, chest depth (CD), abdominal diameter (AD), umblical cord diameter (UCD), ruminal length (RL), placentome diameter (PLD), and kidney length (KL) increased continuously till the end of experiment from first day of appearance except placentome which decreased in size after 97 days. The testicular parenchyma was observed to be anechoic at beginning of the development of the testes and it became moderately echogenic as age advanced. However, epididymal echogenicity observed lower as compared to testicular echogenicity. The measurement of testicular length and width revealed that development of left and right testis with age is exactly the same in Harnali lambs and are almost mirror image of each other. The epididymides dimensions increased with advancement of age in direct proportions and a high degree of symmetry was observed in epididymis tail growth. The peak systolic velocity (PSV) and end diastolic velocity (EDV) increased from day 38 to 139 days of gestation. The pulsatile index (PI) and resistive index (RI) of blood flow showed a non-significant decrease throughout the gestation. Similar trend was observed in blood flow parameters (PSV, EDV, PI, RI) to lamb testis as recorded in pregnant ewe. Doppler data regarding blood circulation to umbilicus and testis obtained in the present study might be useful in differentiation of normal and abnormal pregnancy and testicular development however, more studies are needed in this field. 3D scanning of testis showed greater details of internal structure of testis as compared to 2D. From the present study it is concluded that 3D scanning is more useful than 2D scanning to assess developmental changes of fetus in utero and postnatal testis.
