Improved milk production through PG–PL system by provision of in‐house shelter management in lactating Murrah buffaloes during winter season

Comprehensive information on the role of β‐casein and plasminogen–plasmin (PG–PL) system in milk secretion of Murrah buffaloes during winter season is lacking, although effects of cold stress can be ameliorated to an extent by altering microclimate at farm level. Hence, this study was aimed to determine the changes in productivity along with PG–PL system of milk, plasma hormones and metabolites of buffaloes during winter (December–January) season under two different management systems. Average minimum temperature and wind chill index during this season were 7.02 and 12.74 °C respectively. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals were placed in‐house with floor bedding of paddy straw and the control group animals in loose housing system without straw bedding. Physiological responses were recorded, and milk and blood samples were collected at weekly intervals for six‐week experimental period. Under in‐house management system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as respiration rate and pulse rate were significantly reduced (p < 0.01) as compared to the control, which subsequently resulted higher milk yield by 9.92% (p < 0.05). Analysis of milk samples revealed higher concentration of plasminogen (10.6 vs. 8.05 μg/ml; p < 0.01) and β‐casein (p < 0.05), and lower plasmin level (0.299 vs. 0.321 μg/ml; p < 0.05) in buffaloes under treatment group. It was also found that plasma cortisol, glucose and non‐esterified fatty acids levels were higher (p < 0.01) in control group as compared to the treatment animals by 13.6%, 8.14% and 12.6% respectively. However, milk composition, growth hormone, epinephrine and norepinephrine level in plasma were similar in both the groups. Hence, it may be concluded that provision of in‐house shelter management with floor bedding of paddy straw during winter was effective to minimize environmental stress and improved milk production through manipulation of PG–PL system in buffaloes.     

Dominant follicles development and estradiol‐17β concentrations in non‐ovulating and ovulating post‐partum Bulgarian Murrah buffaloes

This study was designed to evaluate the dominant follicles development and the estradiol‐17β concentrations in non‐ovulating and ovulating post‐partum buffaloes. Sixteen Bulgarian Murrah buffaloes were submitted to transrectal ultrasonographic examination from the 1st post‐partum day until day 50, 3 days apart. The follicular diameter of the different categories of follicles and the ovulations was recorded. The animals were allocated into two groups: I (n = 6) non‐ovulating and II (n = 10) ovulating buffaloes. Serum estradiol‐17β concentrations on the days for dominant follicle registration were measured by enzyme‐linked immunosorbent assay. The results were statistically processed by analysis of variance, non‐parametric and correlation analysis. The mean intervals between calving and first dominant follicle detection differed significantly (p < .05) among the groups (19.5 ± 6.2 vs. 13.8 ± 5.1 days), while the mean intervals between registered dominant follicles from two successive waves were comparable. The mean follicular diameters for the same category follicles in both groups were similar. Different estradiol‐17β concentrations (p < .05) for the first dominant follicle between non‐ovulating (23.5 ± 7.0 pg/ml) and ovulating (33.3 ± 8.4 pg/ml) buffaloes were determined. The cumulative percentages of buffaloes with firstly detected dominant follicle and ovulating animals correlated positively (r ≥ .84; p < .05) to post‐partum days. In conclusion, non‐ovulating and ovulating post‐partum Bulgarian Murrah buffaloes showed differences in the development of the first dominant follicle and estradiol‐17β concentrations during the time of dominant follicles detection.     

Effect of pre‐partum supplementation of vitamin E to Murrah buffaloes on immune functions and viability of calves

This study was undertaken in buffalo neonates born to vitamin E (dl ‐ alpha ‐ tocopherol acetate) ‐ supplemented and non‐supplemented Murrah buffaloes. Calves from vitamin E‐supplemented buffaloes (n = 10; vitamin E ‐supplemented calves [VeC]) and non‐supplemented buffaloes (n = 10; control calves [CC]) constituted the treatment and control groups respectively. Two colostrum samples were taken at the first post‐partum milking and again after 12 h from dams for IgG estimation. Sampling of blood was performed on days 0 (before colostrum feeding), 1, 3, 7, 14, 21, 28, 42, 56, 70, 84, 98, 112 and 126 post‐birth and analysed for apparent efficiency of absorption (%) of IgG and various immune parameters. Colostral IgG level was significantly higher (p < 0.05) in vitamin E‐supplemented buffaloes. The calves in both groups were born hypogammaglobulinemic with IgG level <5 g/l. However, first colostrum feeding resulted in significantly elevated IgG levels (>10 g/l) in calves of both groups at 24 h, which remained high afterwards. Apparent efficiency of absorption (%) of IgG at 24 h was significantly higher (p < 0.05) in VeC than in CC. Plasma Nitric Oxide (NO) levels were significantly elevated in the calves of either group at birth, which declined significantly (p < 0.01) afterwards. Vitamin E feeding to dams had no added effect on NO levels in experimental calves. Total leucocyte counts did not differ significantly between the two groups. However, lymphocyte and neutrophil counts changed significantly between groups (p < 0.01) and days (p < 0.01), with lymphocytes increasing and neutrophils declining with age. This study revealed that the calves were immunologically immature at birth. Ante‐partum supplementation of vitamin E did not influence plasma NO or IgG but had a significant effect on colostral IgG (p < 0.05). It also improved the apparent efficiency of absorption (%) of IgG at 24 h in VeC as compared to CC.     

Relationship of temperament with oestrous behaviour,resumption of ovarian cyclicity and milk yield in post‐partum Murrah buffaloes

This study was designed to investigate the relationship of temperament with oestrus, resumption of ovarian cyclicity and milk yield in post‐partum buffaloes. For this study, 102 post‐partum Murrah buffaloes were observed for temperament in open paddock and during milking. Based on the temperament score (1–5 score), they were classified into docile, slightly restless, restless, aggressive and nervous. Oestrous behaviour was monitored twice daily in all the buffaloes along with milk yield (weekly), body weight (BW) and body condition score (BCS) at day 0, 30, 60 and 90 post‐partum. Based on the temperament score, the number of buffaloes classified as docile, slightly restless, restless and aggressive was 37 (36.27%), 28 (27.45%), 30 (29.41%) and 7 (6.86%), respectively, but none under nervous category. Number of buffaloes resuming cyclicity in various temperaments groups were as follows: docile (n = 35; 94.59%), slightly restless (n = 22; 78.57%), restless (n = 20; 66.67%) and aggressive (n = 7; 85.71%). Temperament was not correlated (p = .128; r=?.152) with oestrous behaviour. But, temperament was correlated (p < .0001; r = .384) with the resumption of cyclicity as docile and slightly restless buffaloes resumed cyclicity (p < .0016) earlier. Temperament of buffaloes was weakly correlated (p = .0017; r=?.306) with milk production with higher (p < .0358) total milk yield in docile (1022 ± 23.75 kg) and slightly restless buffaloes (948 ± 35.86 kg) as compared to other temperament groups. In summary, temperament of post‐partum buffaloes was related with resumption of ovarian cyclicity and milk yield, but not oestrous behaviour.     

Evaporative cooling in late gestation heat‐stressed Murrah buffaloes increases efficiency of next reproductive cycle

Evaporative cooling during late gestation period improves post‐partum reproductive performance in Murrah buffaloes. To prove this hypothesis, sixteen pregnant dry Murrah buffaloes at sixty days pre‐partum were selected and divided into two groups of eight animals each. Group 1 of buffaloes (Cooled/CL) was managed under fan and mist cooling during dry period, whereas second group of buffaloes (non‐cooled/NCL) remained without the provision of cooling. After parturition, all the animals were managed under evaporative cooling till the end of experimental period. Reproductive performance in cooled (CL) and non‐cooled (NCL) groups, respectively, viz. 1st and 2nd ovulation from calving (48.63 ± 2.41, 69.25 ± 2.34 days and 57.75 ± 3.35, 93.63 ± 2.84 days); calving to conception interval (117.88 ± 4.21 days and 117.88± 4.21 days); conception rate (87.5% ± 2.16% and 57% ± 2.26%); and follicular diameter at the time of 1st and 2nd ovulation (14.84 ± 0.16, 15.75 ± 0.13 mm and 12.65 ± 0.13, 13.35 ± 0.11 mm) varied significantly (p < .05). Total peak oestrogen concentration was significantly (p < .05) higher in cooled (26.7 ± 1.32 pg/ml) relative to non‐cooled (20.7 ± 1.22 pg/ml) buffaloes. Time from onset of oestrus to ovulation varied significantly (p < .05) in cooled (32 ± 2.22 hr) and non‐cooled (40 ± 2.86 hr) buffaloes. The peak progesterone concentration reached to (4.25 ng/ml) in cooled group and (4.16 ng/ml) in non‐cooled group after first ovulation.     

Ovarian responses of dairy buffalo cows to timed artificial insemination protocol,using new or used progesterone devices,during the breeding season (autumn–winter)

This study evaluated the effect of new or used P4 devices on the ovarian responses of dairy buffalo that were administered an estradiol (E2) plus progesterone (P4)‐based timed artificial insemination (TAI) protocol during the breeding season. On the first day of the TAI protocol, 142 cows were randomly assigned to receive one of the following: a new device (New; 1.0 g of P4; n = 48); a device that had previously been used for 9 days (Used1x, n = 47); or a device that had previously been used for 18 days (Used2x, n = 47). Ultrasound was used to evaluate the following: the presence of a corpus luteum (CL); the diameter of the dominant follicle (ØDF) during protocol; ovulatory response; and pregnancies per AI (P/AI). Despite similar responses among the treatments, there was a significant positive association of the ØDF during TAI protocol with ovulatory responses and number of pregnancies. In conclusion, satisfactory ovarian responses and a satisfactory pregnancy rate were achieved when grazing dairy buffalo were subjected to the TAI protocol in breeding season, independent of whether a new or used P4 device was used. Furthermore, the presence of the larger follicle was associated with a higher ovulation rate and higher P/AI following TAI.     

Random regression models to estimate genetic parameters for test‐day milk yield in Brazilian Murrah buffaloes

The objective of this work was to estimate covariance functions for additive genetic and permanent environmental effects and, subsequently, to obtain genetic parameters for buffalo’s test‐day milk production using random regression models on Legendre polynomials (LPs). A total of 17 935 test‐day milk yield (TDMY) from 1433 first lactations of Murrah buffaloes, calving from 1985 to 2005 and belonging to 12 herds located in São Paulo state, Brazil, were analysed. Contemporary groups (CGs) were defined by herd, year and month of milk test. Residual variances were modelled through variance functions, from second to fourth order and also by a step function with 1, 4, 6, 22 and 42 classes. The model of analyses included the fixed effect of CGs, number of milking, age of cow at calving as a covariable (linear and quadratic) and the mean trend of the population. As random effects were included the additive genetic and permanent environmental effects. The additive genetic and permanent environmental random effects were modelled by LP of days in milk from quadratic to seventh degree polynomial functions. The model with additive genetic and animal permanent environmental effects adjusted by quintic and sixth order LP, respectively, and residual variance modelled through a step function with six classes was the most adequate model to describe the covariance structure of the data. Heritability estimates decreased from 0.44 (first week) to 0.18 (fourth week). Unexpected negative genetic correlation estimates were obtained between TDMY records at first weeks with records from middle to the end of lactation, being the values varied from ?0.07 (second with eighth week) to ?0.34 (1st with 42nd week). TDMY heritability estimates were moderate in the course of the lactation, suggesting that this trait could be applied as selection criteria in milking buffaloes.     

Histological and immunohistochemical investigations of ovarian interstitial glands during non‐breeding season in camels (Camelus dromedarius)

The aim of this was to investigate the histology and immunohistochemistry of interstitial glands during non‐breeding season in camel ovaries. A total of 21 mature, non‐pregnant and apparently healthy camels aged between 8 and 12 years were slaughtered. The ovaries were removed within 15 min, cleaned from adipose tissue, weighted and examined grossly. The histological preparation was made, and then, the blocks were cut at 3–5 microns thickness and stained by H&E for histological examinations. Moreover, some sections were stained with Sudan Black for lipid detection. Immunohistochemical analysis of paraffin‐embedded ovarian tissues was performed to detect the localization of S‐100, vimentin, progesterone receptors (PR) and oestrogen receptors (ER). Immunoreactive signals were detected using UltraVision Detection System. The results revealed that the interstitial glands were located in the cortical region and they were arranged in various arrangements either single, in couple or in groups rich in lipid droplet. All interstitial gland arrangements were enclosed by connective tissue capsules containing fibroblasts and collagenous fibres separated them from the surrounding ovarian structures. Both interstitial glands and their surrounding CT were penetrated by several blood vessels. There was a strong immunoreactive signal for S‐100 in the nuclei of interstitial cells, and no signals were detected either in cells of the interstitial glands or their connective tissue with PR. We could conclude that the interstitial gland is distinct in ovary of camel and further studies are needed to elucidate its rule in steroid synthesis.     

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.     

Effects of mannan‐oligosaccharides and Lactobacillus acidophilus supplementation on growth performance,nutrient utilization and faecal characteristics in Murrah buffalo calves

A study of 120 days was undertaken to ascertain the effect of mannan‐oligosaccharides (MOS) and Lactobacillus acidophilus supplementation on growth performance, nutrient utilization and faecal characteristics in Murrah buffalo calves. Twenty Murrah buffalo calves of 5–7 days old and 31 ± 2.0 kg of body weight (BW) were randomly assigned into four groups. Group I served as the control (CON) in which only basal diet (concentrate mixture and green fodder) was provided, without any supplementation. Mannan‐oligosaccharides at 4 g/calf/day were supplemented as prebiotic to Group II (PRE), whereas Group III (PRO) received Lactobacillus acidophilus in the form of fermented milk as probiotic at 200 ml/calf/day having 10⁸ CFU/ml and Group IV (SYN) was supplemented with both MOS and Lactobacillus acidophilus as synbiotic at similar dose. Final BW (kg), dry matter intake, average daily gain, feed conversion efficiency and structural growth measurements were improved (p < .05) in the treatment groups compared to control. Digestibility of neutral detergent fibre was higher (p < .05) in SYN followed by PRE and PRO than control. The faecal lactobacilli and bifidobacterium population was higher (p < .05) in all the supplemented groups with a concomitant reduction in faecal coliform count as compared to control. Faecal ammonia, lactate and pH were also altered favourably (p < .05) in all the supplemented groups as compared to CON. The faecal volatile fatty acids were higher (p < .05) in PRE, PRO and SYN group than CON. The incorporation of MOS and Lactobacillus acidophilus in diet either individually or in combination as synbiotic has the potential to improve the performance and faecal characteristics in Murrah buffalo calves; however, the observed responses among the treatment groups were more evident in the synbiotic fed group compared to individual supplementation of MOS and Lactobacillus acidophilus.     

Effectiveness of controlled internal drug release device treatment to alleviate reproductive seasonality in anestrus lactating or dry Barki and Rahmani ewes during non‐breeding season

This study aimed to evaluate the effectiveness of hormonal treatments on ovarian activity and reproductive performance in Barki and Rahmani ewes during non‐breeding season. Forty‐eight multiparous ewes, 24 Barki and 24 Rahmani ewes were divided into two groups, 12 lactating and 12 dry ewes for each breed. Controlled internal drug release (CIDR) device was inserted in all ewes for 14 days in conjunction with intramuscular 500 IU equine chronic gonadotrophin (eCG) at day of CIDR removal. Data were analysed using PROC MIXED of SAS for repeated measures. Breed, physiological status and days were used as fixed effects and individual ewes as random effects. Barki ewes recorded higher (p < .05) total number of follicles, number of large follicles, serum estradiol concentration and estradiol: progesterone (E₂:P₄) ratio compared to Rahmani ewes. Lactating ewes recorded higher (p < .05) number of small follicles and lower concentration of total antioxidant capacity (TAC) compared to dry ewes. Number and diameter of large follicles recorded the highest (p < .05) values accompanied with disappearance of corpora lutea at day of mating. Serum progesterone concentration recorded lower (p < .05) value at day of mating and the highest (p < .05) value at day 35 after mating. CIDR‐eCG protocol induced 100% oestrous behaviour in both breeds, but Rahmani ewes recorded longer (p < .05) oestrous duration compared to Barki. Conception failure was higher (p < .05) in Barki compared to Rahmani ewes. In conclusion, CIDR‐eCG protocol was more potent in improving ovarian activity in Barki compared to Rahmani ewes, but this protocol seems to induce hormonal imbalance in Barki ewes that resulted in increasing conception failure compared to Rahmani ewes.     

Crystalline amino acids supplementation improves the performance and carcass traits in late‐finishing gilts fed low‐protein diets

This study investigated the effects of amino acids (AA) supplementation in low‐crude protein (CP) diets on the growth performance and carcass characteristics in late‐finishing gilts. Ninety gilts (93.8 ± 5.5 kg) were randomly allotted to one of the five diets which consisted of a normal‐CP (137 g/kg) or four low‐CP (105 g/kg) diets for 28 days. The low‐CP diets were supplemented with lysine + threonine + methionine (LCM), LCM + tryptophan (LCT), LCT + valine (LCV) or LCV + isoleucine (LCI), respectively. Non‐significant difference in average daily gain (ADG) was obtained in gilts receiving the control and LCV diet, which was higher than that of gilts fed the LCM diet (p < .05). The additions of crystalline AA in the low‐CP diet resulted in the improvements in ADG (linear and quadratic effect, p < .05) and fat‐free lean gain (quadratic effect, p < .05) and influenced the valine concentration (linear and quadratic effect, p < .05) and proportion of saturated fatty acid (linear effect, p < .05) in longissimus muscle. The results indicated that the valine supplementation could further improve the performance in 94 to 118 kg gilts fed the 105 g/kg CP diet.     

Monitoring of libido and semen quality parameters in melatonin‐treated French alpine bucks during the non‐breeding season

The aim of this study was to investigate the effects of exogenous melatonin on libido and semen quality parameters in bucks during the non‐breeding season. Twelve bucks of the French alpine breed from 1.5 to 4 years of age were assigned into melatonin (MG) and control (CG) groups, with 6 bucks in each group. The experimental period was 3 months (March–May), divided into six periods of 15 days each. The bucks in the MG group received four melatonin implants at the end of March. Two semen samples were taken from the bucks by artificial vagina once per week and their libido estimated. Volume and spermatozoa concentration, their mass motility and motility, proportion of live and total abnormal and forms with abnormal head and tail were determined in the obtained ejaculate samples. The total number of spermatozoa and functional spermatozoa fraction in the ejaculate was also calculated. The MG bucks had significantly higher mass motility and motility of spermatozoa in the first half of April, and a higher proportion of live spermatozoa in the first and second half of April (p < .05). Differences in libido intensity were not significant. The results indicated that the application of melatonin significantly improved the qualitative parameters of semen in bucks, as seen in increased mass motility, motility of spermatozoa and proportion of live spermatozoa shortly following melatonin insertion. Therefore, the results of the current study are novel regarding the use of melatonin treatment during the non‐breeding season to improve the qualitative parameters of ejaculates in bucks.     

Microscopic detection of IgY‐Fc binding signal in the inner layers of ovarian follicular tissue in quail

In avian species, it has been assumed that an Fc receptor in the ovarian follicles mediates immunoglobulin Y (IgY) transport into the yolk. However, no such receptor responsible for IgY has been identified to date. To examine potential IgY binding activity in the entire ovarian follicle, whole‐mount sections of quail ovarian follicle were incubated with the Fc fragment of chicken IgY (cIgY). Whole‐mount frozen sections of the second largest ovarian follicle were prepared, and then the sections were incubated with digoxigenin‐labeled Fc or Fab fragments of cIgY. Microscopic observation revealed that incubation with the cIgY‐Fc fragment produced a binding signal in the inner layer of the ovarian follicular tissues, most likely in the granulosa cell layer. However, no such signal was detected when the sections were incubated with cIgY‐Fab. Coincubation of the ovarian sections with Alexa488‐labeled cIgY‐Fc and antiserum raised against ZP1, an envelope protein specifically localized in the perivitelline layer, demonstrated that the source of the Fc binding signals partly coincided with the perivitelline layer. In conclusion, our data suggest that potential IgY binding substances interacting with the Fc domain are present in the inner layers of ovarian follicular tissues, most likely in the granulosa cell layer and/or in the perivitelline layer.     

Assessment of pregnancy‐associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera

Pregnancy‐associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)‐PAG systems: RIA‐1 (antiserum raised against bovine PAG67kDa; AS#497), RIA‐2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA‐3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA‐2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA‐2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA‐3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA‐1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG‐RIA systems to measure PAG concentration in swamp buffalo samples.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Growth differentiation factor‐9 improves development,mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

This study analysed the effect of growth differentiation factor‐9 (GDF‐9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α‐MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α‐MEM⁺—control medium) or α‐MEM⁺ supplemented with 1, 10, 50 or 100 ng/ml GDF‐9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF‐9 than other treatments, except for 10 ng/ml of GDF‐9 (p > 0.05). Treatment containing 100 ng/ml GDF‐9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF‐9 showed more oocytes in MI than α‐MEM⁺, 1 or 50 ng/ml GDF‐9 (p < 0.05). In conclusion, 100 ng/ml GDF‐9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.