Development and Application of a Sensitive, Second Antibody Format Enzymeimmunoassay (EIA) for Estimation of Plasma FSH in Mithun (Bos frontalis).

Mithun (Bos frontalis) is a semi-wild rare ruminant species. A simple sensitive enzymeimmunoassay suitable for assaying FSH in the blood plasma of mithun is not available which thereby limits our ability to understand this species reproductive processes. Therefore, the aim of this article was to develop a simple and sensitive enzymeimmunoassay (EIA) for estimation of FSH in mithun plasma and apply the assay to understand the estrous cycle and superovulatory process in this species. To accomplish this goal, biotinylated FSH was bridged between streptavidin-peroxidase and immobilized antiserum in a competitive assay. Forty microlitre mithun plasma was used directly in the EIA. The FSH standards were prepared in hormone free plasma and ranged from 5-1280 pg/well/40 µL. The sensitivity of EIA was 5 pg/well FSH, which corresponds to 0.125 ng/mL plasma and the 50% relative binding sensitivity was 90 pg/well/40 µL. Although the shape of the standard curve was not influenced by different plasma volumes viz. 40 and 80 µL, a slight drop in the OD450 was observed with the increasing volume of plasma. Parallelism tests conducted between the endogenous mithun FSH and bovine FSH standards showed good homology between them. Plasma FSH estimated using the developed EIA and commercially available FSH EIA kit in the same samples were correlated (r = 0.98) and showed linearity. Both the Intra- and inter-assay CV were below 6%. Recovery of known concentrations of added FSH showed linearity (r = 0.99). The developed EIA was further validated biologically by estimating FSH in cyclic cows for the entire estrous cycle, in mithun heifers administered with GnRH analogues and in mithun cows during superovulatory treatment with FSH. In conclusion, the EIA developed for FSH determination in mithun blood plasma is simple and highly sensitive for estimation of mithun FSH in all physiological conditions.

Effect of supplementation of vitamin E, copper and zinc on the in vitro phagocytic activity and lymphocyte proliferation index of peripartum Sahiwal (Bos indicus) cows.

To study the effect of vitamin E (VE), copper (Cu) and zinc (Zn) supplementation on the in vitro phagocytic activity (PA) and lymphocyte proliferation response (LPR) of blood neutrophils and lymphocytes, thirty Sahiwal pregnant cows (six in each group) in their late gestation at 30 days before the expected date of calving were selected from the NDRI experimental herd and supplemented with various micronutrients from 30 days before calving to 45 days after calving. Cows were supplemented individually with VE (1000 IU/cow/day), Cu (20 ppm/cow/day) and Zn (80 ppm/cow/day) and also with a combination of VE, Cu and Zn to study cumulative effect of all micronutrients. One group without any supplementation acted as a control. Blood neutrophils and lymphocytes were isolated and studied for their PA and LPR. Supplementation of micronutrients like VE, Cu, Zn and a combination of all these nutrients significantly (p < 0.01) increased the PA of experimental cows as compared to control (unsupplemented) cows during the pre-partum period. During post-partum, all the micronutrients (VE, Cu, Zn and their combination) showed a significant (p < 0.01) increase in the PA of experimental cows as compared to control cows. Of all the groups, significant (p < 0.01) and maximum PA was observed in the combination group followed by Zn-supplemented group during both the pre- and post-partum period. A significant (p < 0.01) increase in LPR of B lymphocytes was observed in combination-supplemented group during the pre-partum period and during both the pre- and post-partum period in the Cu-supplemented group.

The endocrine changes, the timing of ovulation and the efficacy of the Doublesynch protocol in the Murrah buffalo (Bubalus bubalis).

Experiments were conducted to investigate (a) the timing of ovulation and the associated endocrine changes (progesterone, estrogen and LH) during estrous cycle and (b) the efficacy, with respect to the pregnancy rate, in cycling and anestrus in Murrah buffaloes subjected to the Doublesynch protocol during the low breeding season. In experiment 1, 10 cycling buffaloes were administered PGF(2α) on day 0 (without regard to the estrous cycle stage), GnRH on day 2, a second PGF(2α) injection on day 9, and a second GnRH injection on day 11. Transrectal palpation was performed at 2-h intervals after the first and second GnRH treatments until ovulation was detected or for upto 96 h. The plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals starting 2 days before the onset of the protocol and continued until the day of the second detected ovulation. The plasma LH and total estrogen concentrations were measured in blood samples collected at 30-min intervals for 8h following the first and second GnRH injections and thereafter at 2-h intervals until 2h after the detection of ovulation. Ovulation occurred in 9/10 buffaloes (90%) at 22.2 ± 1.2 h (mean ± S.E.M.; range 18.0-26.0 h) and 10/10 buffaloes (100%) at 23.2 ± 1.0 h (mean ± S.E.M.; range 20.0-28.0 h) after the first and second GnRH treatments, respectively. The peak LH concentrations of 99.8 ± 28.5 ng/ml (range 37.8-320.0 ng/ml) and 62.3 ± 11.9 ng/ml (range 20.9-143.9 ng/ml) occurred 2.1 ± 0.3 h (range 1.0-3.5 h) and 2.3 ± 0.3 h (range 0.5-3.0 h) after the first and second GnRH treatments, respectively. The total estrogen concentration gradually increased from the day of both the first and second PGF(2α) administrations until the LH peak (with great variability) and then gradually declined to the basal level, which was reached at the time ovulation was detected. In experiment 2, 10 cycling and 11 non-lactating anestrus buffaloes were subjected to the Doublesynch protocol with timed artificial insemination (TAI) 16 and 24 h after the second GnRH treatment, and 55 cycling buffaloes were inseminated after spontaneous estrus was detected (control group). The pregnancy rates were 60% using TAI on cycling buffaloes (experiments 1 and 2), 55% for anestrus buffaloes (experiment 2), and 27.3% for cycling buffaloes inseminated following spontaneous estrus. The overall pregnancy success rates after the Doublesynch protocol in both cycling and anestrus buffaloes increased by 30.8% compared to spontaneous estrus (58.1% vs. 27.3%). In conclusion, the Doublesynch protocol effectively synchronized ovulation twice (after the first and second GnRH treatments) irrespective of the stage of estrous cycle in Murrah buffaloes. The study also demonstrated that the Doublesynch protocol followed by TAI significantly (P<0.005) enhanced the pregnancy rate in cycling and anestrus buffaloes in comparison to untreated controls during the low breeding season.

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ß-actin (ACTB), ß-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ß-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

Influence of feeding Tinospora cordifolia peripartum on lactation parameters in crossbred cows.

For studying the effect of dietary supplementation of guduchi (Tinospora cordifolia) peripartum on lactation an investigation was conducted on 15 pregnant Karan Fries crossbred cows which were divided into two groups: treatment group of eight cows which were supplemented with guduchi at 60 g/day for 45 days prepartum and 120 g/day for 45 days postpartum; control group of seven pregnant cows which were not supplemented with guduchi. Jugular blood samples were collected from all cows during the periparturient period for analysis of various blood cell and plasma parameters. A significantly higher total leukocyte count, lymphocyte count, neutrophil count and neutrophil/lymphocyte ratio was recorded in the guduchi supplemented treatment group in comparison to untreated control cows throughout the experimental period. The increase of milk production over 305 days of lactation due to guduchi supplementation was significant (p < 0.05). A significant (p < 0.05) reduction in somatic cell count was also observed during the experimental period. Milk composition (fat, protein, lactose and SNF) was similar (p > 0.05) for both the groups. Plasma non esterified fatty acid (NEFA) concentrations were significantly higher (p < 0.01) in cows supplemented with guduchi throughout the course of study. Plasma concentration of growth hormone in the treated cows was also significantly higher beginning on the day of parturition up to 3 weeks postpartum (p < 0.05) in comparison to unsupplemented group.

Temporal changes in circulating levels of plasma interleukin-8 during peripartum period in Murrah buffaloes (Bubalus bubalis) under subtropical climate.

The objective of this study was to elucidate the changes in circulating levels of plasma interleukin-8 (IL-8) during peripartum period in Murrah buffaloes (Bubalus bubalis). IL-8 was estimated in blood plasma of healthy peripartum Murrah buffaloes (n=6) on days ± 30, ± 15, ± 5, ± 3, ± 1 and 0 pre- and postpartum with respect to the day of parturition (day 0) in each of the two different seasons (hot-humid and spring). The mean microclimate Temperature-Humidity Index (THI) during spring season was significantly lower (p<0.01) than the corresponding THI in hot-humid season. In both the seasons, plasma IL-8 remained lower in prepartum period (≤ 46.56 ± 14.08 pg/ml during spring and ≤ 73.18 ± 18.56 pg/ml during hot-humid season) than in the postpartum period (≥ 51.41 ± 13.82 pg/ml during spring and ≥ 84.13 ± 16.97 pg/ml in hot humid season). During spring, the IL-8 levels were significantly higher (P<0.05) on days+5 and +15 postpartum in comparison to the IL-8 levels on days -30, -5, and -3 prepartum. During hot-humid season, IL-8 level was significantly higher (P<0.05) on day +30 as compared to the IL-8 levels on days -30 and -5 prepartum. The correlation between IL-8 and mean microclimate THI was significant (r=0.25, P<0.01). From the results, it is concluded that peripartum period in buffaloes is associated with an inflammatory response leading to significantly higher plasma IL-8 during parturition and postpartum period than in the pre-partum period.

Efficacy of Ovsynch protocol with antiprolactin treatment for timed artificial insemination during non-breeding seasons in yaks (Poephagus grunniens L.).

An attempt was undertaken to investigate the efficacy of Ovsynch protocol for timed artificial insemination (TAI) with or without Norprolac (antiprolactin) treatment during non-breeding season (winter months) in yaks (n = 25). During non-breeding season, plasma prolactin profile has been reported high due to cold and nutritional stress. The Norprolac dose of i.m. administration was standardized for prolactin suppression. Three different doses viz. 2.5, 5.0 and 7.5 mg were attempted and the dose of 7.5 mg Norprolac i.m. per animal was found to be suitable for suppression of prolactin levels up to 30 h. Ovsynch treatment with Norprolac induced more number of oestrous symptoms per animal (4.8 vs 2.1), higher LH peak concentration (24.01 vs 16.16 ng/ml), longer duration of LH surge (6.8 vs 5.2 h) and higher conception rate (70 vs 30%) in Ovsynch plus Norprolac treated animals compared with animals treated with Ovsynch alone. Therefore, this study clearly indicates the opportunity for practical application of the Ovsynch plus Norprolac protocol for TAI in yaks during non-breeding seasons.

Plasma concentrations of 13, 14-dihydro-15-keto-prostaglandin F2alpha, progesterone and cortisol during periparturient period in yaks (Poephagus grunniens L.).

An attempt was made to determine plasma concentrations of, 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM), cortisol and progesterone during periparturient period in yak. Plasma PGFM level showed an increasing trend beginning day 5 prior to parturition (0.48 +/- 0.14 ng/ml) and increased steeply thereafter to reach a peak level (17.16 +/- 1.31 ng/ml) on the day of parturition. The levels, then, declined sharply on day 1 postpartum to reach 1.20 +/- 0.40 ng/ml and thereafter declined gradually over the days to reach 0.28 +/- 0.09 ng/ml on day 20 postpartum and this level was maintained with fluctuation within narrow limits thereafter till conclusion of the blood sampling on day 90 post-calving. The plasma progesterone concentration on days 7 and 6 before parturition was high (2.10 +/- 0.10 and 2.12 +/- 0.10 ng/ml, respectively). The level then decreased gradually and abrupt fall was observed 1-2 days before parturition and became basal on day of parturition (0.24 +/- 0.04 ng/ml). This basal level was maintained till the end of the blood sampling on day 90 postpartum. Plasma cortisol level showed an increasing trend beginning day 2 prior to parturition (2.36 +/- 0.65 ng/ml) and increased steeply thereafter to reach a peak level (26.65 +/- 5.28 ng/ml) on the day of parturition. The levels, then, declined gradually over the days and touched 2.36 +/- 0.25 ng/ml on day 3 postpartum and this level was maintained with fluctuation within narrow limits thereafter till day 7 post-calving.

Changes in endogenous estrogens and expression of behaviors associated with estrus during the periovulatory period in Heatsynch treated Murrah buffaloes (Bubalus bubalis).

The objective of this study was (1) to establish the duration of behavioral estrus signs and timing of ovulation in Murrah buffaloes (n = 10) and (2) to determine relationship between behavioral estrus signs with change in plasma estrogen concentrations in animals treated with Heatsynch protocol. Estrus and its behavioral signs were detected at hourly intervals by visual observations per rectal examination of genitalia and bull parading four times in a day for 30 min each. Among the behavioral signs of estrus, swollen vulva was the best indicator of estrus followed by excitement and chasing by bull (90%). Among the duration of behavioral estrus signs, the first and longest duration of estrus signs was swollen vulva, which was seen up to 21.6 +/- 1.1 h after onset of estrus. The mean total duration of estrus symptoms from appearance to disappearance of all the behavioral estrus symptoms was 40.2 +/- 1.8 h. Endocrine profile during the periestrus period showed that the mean peak concentrations of total estrogen 330.9 +/- 108.3 pg/ml occurred at 9.6 +/- 1.0 h after estradiol benzoate injection. The average number of estrus symptoms observed per animal during Heatsynch treatment was 7.1. Ovulation occurred after 50.0 +/- 2.0 h after estradiol benzoate treatment and 26.7 +/- 2.0 h after end of total estrogen surge, respectively. In conclusion, our results suggest that all signs of behavioral estrus occurred after the preovulatory rise in estrogens. The first sign of estrus was swollen vulva, and this symptom persisted the longest.

Temporal changes in endogenous estrogens and expression of behaviors associated with estrus during the periovulaory period in Murrah buffaloes (Bubalus bubalis).

The objective of this study were (1) to establish the duration of behavioral estrus signs and timing of ovulation in Murrah buffaloes (n = 10) and (2) to determine relationship between behavioral estrus signs with change in plasma estrogen concentrations. Estrus and its behavioral signs were detected at hourly intervals by visual observations, per recta examination of genitalia and bull parading four times in a day for 30 minutes each. Among the behavioral signs of estrus, swollen vulva (80%) was the best indicator of estrus followed by excitement (70%). Among the duration of behavioral estrus signs the first and longest duration of estrus signs was swollen vulva which was seen upto 19.8 +/- 0.8 h after onset of estrus. The mean total duration of estrus symptoms from appearance to disappearance of all the behavioral estrus symptoms was 23.5 +/- 1.7 h. All the behavioral estrus symptoms were observed during the period of estrogen surge. Endocrine profile during the periestrus period showed that the mean peak concentrations of total estrogen 23.9 +/- 3.9 pg/ml occurred at 8.8 +/- 1.7 h after onset of estrus. The average number of estrus symptoms observed per animal during onset of spontaneous estrus was 5.7. Ovulation occurred after 37.4 +/- 1.7 h after onset of estrus and 13.4 +/- 1.0 h after end of total estrogen surge respectively. In conclusion, our results suggest that all signs of behavioral estrus occurred during the preovulatory rise in estrogens. The first sign of estrus to be observed was a swollen vulva and this symptom persisted the longest.

Seasonal variations in plasma glucocorticoid levels in yaks (Poephagus grunniens L.) (Bos grunniens).

The changes of glucocorticoid concentrations were measured by highly sensitive enzymeimmunoassay procedures using second antibody coating technique and cortisol -HRP amplified system for one whole calendar year in yaks. The year was divided into seasons of cool, intermediate and hot seasons. The glucocorticoid values were lower (P < .01) in hotter months than in cool and intermediate seasons. Rectal temperature and respiration rates were indicative of heat stress in the hot temperature season and gave significant negative correlation with circulating corticoid concentration.

Divergent development of testosterone secretion in male zebu (Bos indicus) and crossbred cattle (Bos indicus x Bos taurus) and buffaloes (Bubalus bubalis) during growth.

Delayed pubertal development and low fertility of Bos indicus x Bos taurus crossbred male cattle and domestic buffaloes is hardly understood hence, a sensitive enzymeimmunoassay (EIA) was developed using the second antibody-coating technique and testosterone-3-O-carboxymethyloxime-horseradish peroxidase conjugate as a label for determination of testosterone in blood plasma. The EIA was validated by standard criteria. Blood samples were collected by venipuncture from growing male cattle (Karan Fries and Sahiwal) and buffalo (Murrah) and testosterone was estimated using the EIA procedure. Plasma testosterone concentrations increased significantly (P < 0.05) with advancing age. Testosterone concentrations were significantly (P < 0.01) higher in Sahiwal males in comparison to Karan Fries males. The low testosterone levels in crossbred than Sahiwal could imply that crossbred males have either not stabilized genetically or not adapted well in Indian climatic conditions resulting in poor libido and poor semen quality. The low testosterone levels in Murrah buffalo males may be the possible reason for delayed maturity in this species. The direct, sensitive EIA validated for estimating the plasma testosterone concentration was reliable for studying the testosterone profile in blood plasma of males. The results suggest that there could be a requirement for higher testosterone secretion by males during early stages of growth for attaining early sexual maturity.

Plasma progesterone, oestradiol-17ß and total oestrogen profiles in relation to oestrous behaviour during induced ovulation in Murrah buffalo heifers.

The objectives of this study were to establish the characteristics of oestrous behaviour in Ovsynch (induction of ovulation through administration of GnRH-PGF2-GnRH in a systemic manner on 0, seventh and ninth day respectively) and Ovsynch plus Norprolac (Quinagolide hydrochloride ­ an inhibitor of prolactin secretion) treated Murrah buffalo heifers and to determine the relationships between this behaviour and the plasma concentrations of oestradiol-17ß (E2), total oestrogen, and progesterone. Oestrus was detected by visual observations of oestrus signs, per rectal examination of genitalia and bull parading thrice a day during treatment period. Among all the symptoms, it was observed that bull mounting of heifers in oestrus was highest. Examination of genital tracts per rectum revealed that the cervix was relaxed, uterus was turgid and ovaries had palpable follicle in animals with oestrus. The peak concentrations of E2 (10.81 ± 0.62 pg/ml) and total oestrogen (17.11 ± 1.21 pg/ml) occurred at 9.45 ± 0.85 and 9.64 ± 0.93 h after second GnRH administration, respectively, in Ovsynch treated animals. However, the peak levels of E2 (20.02 ± 2.87 pg/ml) and total oestrogen (32.71 ± 3.15 pg/ml) occurred at 10.18 ± 0.50 and 10.36 ± 0.75 h after second GnRH administration, respectively, in Ovsynch plus Norprolac treated animals. Plasma progesterone concentration was basal (0.20 ± 0.001 ng/ml) during the peri-oestrus period. The plasma progesterone concentration was the lowest on the day of oestrus and increased to register a peak on day 13 ± 2 of the cycle. Oestrous behaviour was positively correlated with the peak concentration of E2 (p < 0.001) and total oestrogen (p < 0.001) during the peri-oestrus period. Inhibition of prolactin by Norprolac administration significantly increased the concentration of E2 and total oestrogen during oestrus in buffaloes in comparison to those recorded in animals subjected to Ovsynch protocol alone. In conclusion, our results suggest that the peak concentrations of E2 and total oestrogen and mean level of E2 and total oestrogen during the peri-oestrus period are the important factors contributing the behavioural manifestation of oestrus in buffalo cows.

Application of sensitive enzymeimmunoassay for determination of testosterone in blood plasma of yaks (Poephagus grunniens L.).

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of testosterone in blood plasma of yaks on microtitreplates using second antibody coating technique and testosterone-horseradish peroxidase as a label has been developed for the first time. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20 mICROl of plasma after 1:10 dilution with assay buffer. The testosterone standard curve ranged from 0.2 to 200 pg/well. The sensitivity of the assay was 0.20 pg/well. Testosterone standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous testosterone. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 5.24 and 8.54%, respectively. Recovery of known concentrations of added testosterone in charcoal stripped plasma was linear (r=0.98). For biological validation of testosterone enzymeimmunoassay, the blood samples were collected from yak cows at -2h before and thereafter at 2h interval until 24h. after gonadotropin releasing hormone (GnRH) administration. There was a rapid increase (p<0.01) of luteinizing hormone (LH) and testosterone 2 and 6h after GnRH administration. Plasma testosterone concentration in normal adult yak bulls was found to be 0.52+/-0.09 ng/ml. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of testosterone directly in yak plasma.

Efficacy of Heatsynch protocol for induction of estrus, synchronization of ovulation and timed artificial insemination in yaks (Poephagus grunniens L.).

The objective of this study was to test the efficacy of induction of estrus, synchronization of ovulation and timed artificial insemination in anestrous yaks using the Heatsynch protocol. In Experiment 1, 10 anestrous yaks were administered an analogue of gonadotropin releasing hormone (GnRH) followed by prostaglandin (PG)F2alpha 7 days later and then estradiol cyponate (ECP) 24 h after that. Ovulation was detected by rectal palpation at 2h intervals beginning at the initial signs of estrus. Blood samples were collected at 2h intervals beginning at the time of ECP injection up to 2h after the occurrence of ovulation for the determination of LH and progesterone. All the animals responded to the Heatsynch protocol with expression of estrus and synchronization of ovulation. The mean time interval from the ECP injection to ovulation was 59.4+/-2.62 h (range 50-72 h). The interval from the LH peak to ovulation was 30.2+/-2.3 h. The high degree of synchrony in ovulation could be attributed to the synchrony in the timing of LH peaks. In Experiment 2, 10 anestrous yaks were treated with the Heatsynch protocol (as in Experiment 1) and TAI was performed at 48 and 60 h after the ECP treatment. Concurrently, 16 cycling yaks were inseminated approximately 12 h after detection of spontaneous estrus. Pregnancy rates were similar in both groups, 40% for TAI and 43.75% for yaks inseminated following spontaneous estrus (p>0.05). From this study, two conclusions can be drawn. First, the Heatsynch protocol can be successfully used to induce and synchronize estrus in anestrous yaks and, second, ovulation following the Heatsynch protocol is synchronized adequately to permit the use of fixed time AI in this species.

Assessment of superovulatory responses in terms of palpable corpora lutea and embryo recovery using plasma progesterone in yaks (Poephagus grunniens L.).

Plasma progesterone profiles were used to assess superovulatory responses in cyclic yaks (n=10) in terms of the number of ovulations and the number of embryos recovered. The animals were synchronized into oestrus following Ovsynch treatment. All the animals received a total of 200 mg Folltropin divided into morning and evening and spread over 4 days, beginning on day 10 of the oestrus cycle (day of expected oestrus=day 0). Plasma samples for progesterone estimation were collected daily starting from the day of expected synchronized oestrus to the day of flushing. All the animals were palpated per rectum on the day of flushing in order to record the number of corpora lutea. Of an estimated 27 ovulations from the nine yaks, only 16 embryos were recovered. Plasma progesterone profiles from individual yaks suggested that a poor superovulatory response in terms of embryo recovery in some animals was caused by the lysis of corpora lutea before flushing which was carried out 7 days after superovulatory oestrus. It was suggested that flushing 5 days post superovulatory oestrus could improve the superovulatory response in this species.

An update on reproduction in yak and mithun.

Yak and mithun are two domesticated herbivores of high economic importance to the farming community living in highlands. Improved yak and mithun production could significantly enhance the living standards of these highlanders. Over the years, their dwindling numbers have been a cause of serious concern. In view of the lack of knowledge on the reproductive physiology of these ruminants, studies have been undertaken to investigate their reproductive endocrinology in recent years. This paper attempts to present the latest information on the endocrine changes associated with their various reproductive processes viz. growth and puberty, oestrous cyclicity and oestrous behaviour, ovulation, and, pregnancy and parturition. The paper also provides the recent developments on research done towards the enhancement of yak and mithun reproductive efficiencies through endocrine and embryo biotechniques.

Estimation of salivary and serum alkaline phosphatase level as a diagnostic marker in type-2 diabetes mellitus with periodontal health and disease: A clinico-biochemical study.

INTRODUCTION: Chronic periodontitis is a multifactorial disease resulting in the inflammation and destruction of the supporting structures around the teeth, leading to tooth mobility and subsequent loss of tooth. Metabolic disorders, such as diabetes mellitus, play a crucial role in the progression of periodontal inflammatory conditions. Alkaline phosphatase (ALP) enzyme plays a key role in gingival inflammation and bone resorption. Hence, the aim of the present study is to compare the serum and salivary alkaline phosphatase levels in chronic periodontitis patients with or without type-2 diabetes mellitus. MATERIALS AND METHODS: A total of 45 individuals were included in the study and divided into three groups: Group I (healthy individual), Group II (Chronic periodontitis without diabetes mellitus type-2) and Group III (Chronic periodontitis with type-2 diabetes mellitus) on the basis of clinical, radiographic and blood sugar examination. The serum and unstimulated saliva were collected from all patients in aseptic condition and samples were analyzed for alkaline phosphatase level using AVANTOR™ Benesphera ALP Kit by fully automated analyzer. RESULTS: The result showed that the concentration of serum and salivary alkaline phosphatase increases significantly in patients with chronic periodontitis with type-2 diabetes mellitus than chronic periodontitis without diabetes mellitus and healthy patients. CONCLUSION: We can conclude that alkaline phosphatase can be used as a key inflammatory diagnostic biomarker in periodontal diseases.

Development and validation of a highly sensitive economic enzymeimmunoassay for prolactin determination in blood plasma of mithun (bos frontalis) and its application during milk let down and cyclicity.

The objective of the present study was to develop and validate highly sensitive and economic enzymeimmunoassay (EIA) for prolactin determination in mithun blood plasma on microtitreplates using the biotin-streptavidin amplification system and second antibody coating technique and to apply this procedure during milk let down and cyclicity in mithuns (Bos frontalis), a semi-wild ruminant. Biotin was coupled to prolactin and used to bridge between streptavidin peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 50 microl mithun plasma. The sensitivity of the EIA procedure was 0.1 ng/ml plasma. Plasma volumes viz., 12.5, 25 and 50 microl did not influence much the shape of standard curve though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma prolactin with bovine prolactin standards used in the assay. It showed good parallelism with the bovine standard curve. Plasma prolactin was estimated in six cyclic mithun cows during an estrous cycle. Mean plasma prolactin concentrations around the day of estrus were recorded to be higher than any other day of the cycle. Prolactin profiles were also obtained in three mithuns before, during and after milking. A sharp release of prolactin shortly after udder stimulation was observed. High levels of prolactin were maintained during milking, falling sharply thereafter. In conclusion, the EIA developed for prolactin determination in mithun blood plasma is sufficiently reliable, economic and sensitive enough to estimate prolactin in all physiological variation in mithun.

Development and validation of a simple, sensitive enzyme immunoassay (EIA) for quantification of prolactin in buffalo plasma.

A simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for prolactin quantification in buffalo plasma (on a microtitreplate) using the biotin-streptavidin-peroxidase amplification and immobilized antiserum in a competitive assay. Prolactin standards (range: 5-5000 pg/(well 50 microL)) were prepared in hormone-free plasma collected from minimal stress non-lactating buffalo heifers in temperate weather. The sensitivity of the EIA procedure was 5 pg/(well 50 microL) (corresponds to 0.1 ng/mL plasma); the 50% relative binding sensitivity occurred at 160 ng/(well 50 microL). Plasma volumes for the EIA, viz. 12.5, 25, and 50 microL, did not influence the shape of standard curve. A parallelism test was carried out to compare the endogenous buffalo plasma prolactin with bovine prolactin standard. To validate the assay biologically, 11 Murrah buffaloes were given a third-generation antiprolactin (Norprolac; 10 mg/animal, i.m.). Blood samples were collected 1 d prior to the start of Norprolac administration and continued up to seventh day in an Ovsynch treatment program. In all animals, there were abrupt declines in prolactin concentrations following Norprolac treatments, which confirmed the biological validation of the EIA. After development and validation of EIA procedure, the concentration of plasma prolactin was determined efficiently in samples collected during both summer and winter samples.