Kisspeptin‐10 stimulates the release of luteinizing hormone and testosterone in pre‐ and post‐pubertal male goats

The aims of the present study were to clarify the effect of kisspeptin‐10 (Kp10) on the secretion of luteinizing hormone (LH) and testosterone (T) in pre‐pubertal and post‐pubertal male ruminants. Four male goats (Shiba goats) were given an intravenous (i.v.) injection of Kp10 (5 µg/kg body weight (b.w.)), gonadotoropin‐releasing hormone (GnRH, 1 µg/kg b.w.), or 2 mL of saline as a control at the ages of 3 (pre‐pubertal) and 6 (post‐pubertal) months. A single i.v. injection of Kp10 significantly stimulated the release of LH and T in both groups. The area under the response curve (AUC) of LH for a 60‐min period after the i.v. injection of Kp10 was significantly greater in the pre‐pubertal goats (P < 0.05). The AUC of T for a 120 min period post‐injection did not differ between the two age groups. A single i.v. injection of GnRH also significantly stimulated the release of LH and T in both groups (P < 0.05). The secretory pattern of LH and T in response to GnRH resembled that in response to Kp10. These results show that the LH‐releasing response to Kp10 is greater in pre‐pubertal than post‐pubertal male goats. They also show that Kp10, as well as GnRH, is able to stimulate the release of T in male goats.     

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.     

Orally administered Chrysin improves post‐thawed sperm quality and fertility of rooster

Chrysin is a bioflavonoid compound found in passion flower, chamomile, propolis and honey at high levels. Post‐thawed sperm quality and fertility of Chrysin‐fed roosters were assessed in this study. Twenty 40‐week‐old male broiler breeders were randomly divided into four groups and fed basal diet supplemented with different levels of Chrysin including 0 (Ch‐0), 25 (Ch‐25), 50 (Ch‐50) or 75 (Ch‐75) mg/day for 12 consecutive weeks. Semen samples were weekly collected from 6th to 9th week of experiment to evaluate some sperm quality parameters including total and progressive motility, plasma membrane integrity and functionality (in fresh and post‐thawed samples) and mitochondrial activity (only in post‐thawed samples). Also, collected semen samples from 10th, 11th and 12th week of experiment were frozen and then artificially inseminated to test fertility rate. According to the results, an improvement in both fresh and post‐thawed sperm quality including total [fresh: 88.00 ± 0.58 and 87.25 ± 0.67 (p < .01); post‐thawed: 51.07 ± 2.05 and 52.72 ± 1.96 (p < .01)] and progressive motility [fresh: 76.00 ± 0.58 and 78.25 ± 0.65 (p < .01); post‐thawed: 40.61 ± 2.01 and 39.88 ± 2.01 (p < .01)], plasma membrane integrity [fresh: 91.60 ± 0.58 and 89.85 ± 0.59 (p < .01); post‐thawed: 56.99 ± 1.86 and 54.39 ± 1.86 (p < .01)] and functionality [fresh: 75.40 ± 0.42 and 77.90 ± 0.96 (p < .01); post‐thawed: 45.69 ± 1.71 and 46.35 ± 1.71 (p < .01)] was noted for both Ch‐50 and Ch‐75, respectively, groups compared to control group. Despite no significant change in mitochondrial activity, fertility rate of post‐thawed spermatozoa was significantly improved in all Chrysin‐fed groups compared to Ch‐0 group. In conclusion, oral Chrysin administration to roosters could ameliorate cryopreservation‐induced impairment of sperm quality and fertility rate.     

Influence of Gonadotrophin‐Induced First Oestrus on Gilt Fertility

The aim of this study was to determine the association between the oestrous response of pre‐pubertal gilts to gonadotrophin injection or boar exposure and their subsequent farrowing rate and litter size. At 154 days of age, randomly selected pre‐pubertal gilts received an intramuscular injection of 400 IU equine chorionic gonadotrophin plus 200 IU human chorionic gonadotrophin (PG600^®; Merck Animal Health; n = 181). From the remaining pool of animals not treated with hormones, the first gilts showing signs of oestrus were selected to act as controls (n = 201). Boar exposure began at 155 days of age for both groups, and gilts were bred at a weight of approximately 130 kg. Comparisons were made between PG600^®‐treated gilts exhibiting oestrus or not within 7 days post‐injection (early and late responders, respectively) and control gilts exhibiting oestrus or not within 30 days after beginning of boar exposure (select and non‐select control gilts, respectively). By 162 days, oestrus was detected in 67.5% of PG600^®‐treated gilts compared with 5.7% of control gilts (p < 0.0001). The proportion of animals observed in oestrus at least three times before breeding was greater for select control gilts compared with early and late responder PG600^®‐treated gilts (p ≤ 0.001). There were no significant differences in farrowing rate and litter size between the four treatment groups. These data indicate that PG600^® is an effective tool to induce an earlier oestrus in gilts, that subsequent farrowing rate and born alive litter size compare favourably to that of select gilts and that gilts failing to respond promptly to hormonal stimulation do not exhibit compromised fertility.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

N‐Acetyl‐d‐galactosamine prevents soya bean agglutinin‐induced intestinal barrier dysfunction in intestinal porcine epithelial cells

Soya bean agglutinin (SBA) is a glycoprotein and the main anti‐nutritional component in most soya bean feedstuffs. It is mainly a non‐fibre carbohydrate‐based protein and represents about 10% of soya bean‐based anti‐nutritional effects. In this study, we sought to determine the effects of N‐Acetyl‐D‐galactosamine (GalNAc or D‐GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC‐J2) cells. The IPEC‐J2 cells were pre‐cultured with 0, 0.125 × 10^?4, 0.25 × 10^?4, 0.5 × 10^?4, 1.0 × 10^?4 and 2.0 × 10^?4 mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre‐incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans‐epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre‐treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin‐3 were lower in the SBA‐treated groups without pre‐treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin‐3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre‐treated groups, occludin and claudin‐3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC‐J2s.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

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.     

Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin‐like growth factor‐1 in pre‐pubertal Holstein heifers

Insulin‐like growth factor‐1 (IGF‐1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR‐SNPs were shown to be related to plasma IGF‐1 concentration in cattle. Hence, the capacity to IGF‐1 production in the liver might be affected by GHR‐SNP and associated with performance in the future. This study examined whether GHR‐SNP is associated with IGF‐1 production in the liver of pre‐pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR‐SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre‐injection than 24 h post‐injection (p < 0.01). Moreover, plasma GH concentrations in AG post‐injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF‐1 concentrations in pre‐injection than post‐injection (p < 0.01), although oestradiol did not change IGF‐1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF‐1 production in the liver of cattle.     

Associations between endotoxin‐induced metabolic changes and temperament in Brahman bulls,

The influence of temperament on the alteration of metabolic parameters in response to a lipopolysaccharide (LPS) challenge was investigated. Brahman bulls were selected based on temperament score. Bulls (10 months; 211 ± 5 kg BW; n = 6, 8 and 7 for Calm, Intermediate and Temperamental groups, respectively) were fitted with indwelling jugular catheters to evaluate peripheral blood concentrations of glucose, blood urea nitrogen (BUN), non‐esterified fatty acids (NEFA), insulin, epinephrine and cortisol before and after LPS administration (0.5 μg/kg BW LPS). Feed intake was also recorded. Intermediate bulls consumed more feed than the Temperamental bulls during the challenge (p = 0.046). Pre‐LPS glucose (p = 0.401) and BUN (p = 0.222) did not differ among the temperament groups. However, pre‐LPS insulin (p = 0.023) was lower, whereas pre‐LPS NEFA (p < 0.001), cortisol (p < 0.001) and epinephrine (p < 0.001) were greater in Temperamental than in Calm and Intermediate bulls. Post‐LPS glucose was increased in Calm and Intermediate bulls but not in Temperamental bulls (p < 0.001). Insulin concentrations post‐LPS were greater in Calm than in Intermediate and Temperamental bulls (p < 0.001). Concentrations of NEFA post‐LPS were greater in Temperamental than in Calm and Intermediate bulls (p < 0.001). Serum BUN concentration increased post‐LPS, with values being greater in Calm and Intermediate than in Temperamental bulls (p = 0.012). Collectively, these data demonstrate that animal temperament is related to the metabolic responses of Brahman bulls following a provocative endotoxin challenge. Specifically, Temperamental bulls may preferentially utilize an alternate energy source (i.e. NEFA) to a greater degree than do bulls of Calm and Intermediate temperaments. The use of circulating NEFA from lipolysis may reduce the negative metabolic consequences of an immune response by allowing for a prompt answer to increasing energy demands required during immunological challenge, compared with the time required for glycogenolysis and gluconeogenesis.     

Evaluation of Follicular Development and Oocyte Quality in Pre‐pubertal Cats

Our study was conducted to assess the follicular development and availability of sound ovarian oocytes for in vitro production (IVP) of embryos in pre‐pubertal cats. The relationship between body and ovarian weight was examined in 93 cats. The results revealed that ovarian weight rapidly increased until 100 days of estimated age. By histological evaluation of ovaries obtained from 11 pre‐pubertal cats with estimated age of <20, 20–40 and 100–120 days, it was clarified that the increase in ovarian weight during kitten growth accompanied the increase in the number and size of antral follicles. The follicular diameter and percentage of normal oocytes in secondary/antral follicles also increased as estimated age (body weight) increased. The oocytes obtained from pre‐pubertal cats with 100–120 days of estimated age were used for IVP of embryos. The results showed that the success rates of in vitro maturation, in vitro fertilization and development to blastocysts after in vitro culture in pre‐pubertal cats were lower than in sexually mature cats. However, the percentage of blastocysts based on the cleaved embryos and cell number of blastocysts in pre‐pubertal cats were comparable to those in mature cats. In conclusion, these results suggest that the ovaries of pre‐pubertal cats with ≥100 days of age contain oocytes with in vitro developmental competence to blastocysts.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Influence of a bovine respiratory disease vaccine with a temperature‐sensitive modified live or killed infectious bovine rhinotracheitis component on oestrous cycle parameters and anti‐Müllerian hormone concentration in nulliparous heifers

The objective of this study was to examine the impact of a bovine respiratory disease complex (BRDC) vaccine with a temperature‐sensitive modified live vaccine (MLV) infectious bovine rhinotracheitis (IBR) component on oestrous cycle parameters and the follicular pool. Twenty‐four Holstein heifers (12.4 ± 0.5 months) previously calfhood vaccinated with an IBR MLV component were enrolled in two replicates (Spring; n = 10 and Fall; n = 14) and were blocked by pre‐vaccination bovine viral diarrhoea (BVD) serum neutralizing (SN) titres. Upon enrolment, heifers were oestrous synchronized with sampling beginning at detected oestrus. At their second heat, heifers were vaccinated with a BRDC calfhood vaccine with a MLV (MLV; n = 12) or killed (K; n = 12) IBR component and sampled for two additional cycles. Serum samples for oestrogen (E2) and progesterone (P4) as well as ultrasound data of ovarian structures were collected every other day. Serum samples for anti‐Müllerian hormone (AMH) were collected at oestrus and mid‐cycle for each cycle, and serum for titres was collected prior to and following vaccination. Data were analysed with the PROC MIXED and GLM procedures of SAS. There was no difference in pre‐ or post‐vaccination titres between MLV and K heifers (p > .5). Vaccination had no impact on P4 concentrations, P4 area under the curve, luteal tissue area, peak E2 production or oestrous cycle length (p > .05). Cycle number did impact AMH concentration (p < .05). In MLV heifers, AMH concentration was highest in cycle 1 (p < .05) while cycles 2 and 3 did not differ (p > .05). This was also true for the K heifers in the Fall replicate (p < .05). Within cycle 2, AMH concentrations were numerically lower between vaccine types (K = 308.22 ± 33.3 pg/ml, MLV = 181.13 ± 32.9 pg/ml; p > .05). Although no differences were seen in overall cycle parameters, differences in AMH concentrations may indicate a reduction of the follicular pool following vaccination and requires further investigation.     

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.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.     

Pharmacokinetics of an extended‐release formulation of eprinomectin in healthy adult alpacas and its use in alpacas confirmed with mange

The study objective was to determine the pharmacokinetics and clinical effects of an extended‐release 5% eprinomectin formulation (Longrange^®) following subcutaneous (s.c.) injection in healthy (n = 6) and mange‐infected (n = 4) adult alpacas. High‐performance liquid chromatography was used to analyze plasma samples obtained at regular intervals for 161 days following a single 5 mg/kg injection s.c. in healthy alpacas, and for 5 days following each dose (3 treatments, 2 months apart) in mange‐affected animals. Skin scrapings and biopsies were performed pre‐ and post‐treatment at two comparable sites in alpacas with mange. Four alpacas served as healthy controls. Eprinomectin plasma concentrations showed a biphasic peak (C_MAX‐1: 5.72 ± 3.25 ng/mL; C_MAX‐2: 6.06 ± 2.47 ng/mL) in all animals at 3.88 ± 5.16 days and 77 ± 12.52 days, respectively. Eprinomectin plasma concentrations remained above 1.27 ± 0.96 ng/mL for up to 120 days. Hematocrit (35.8 vs. 31.3%, P < 0.003) and albumin (3.5 vs. 2.8 g/dL P < 0.006) reduced significantly over 6 months in multidose animals, while fecal egg counts did not differ between groups. Self‐limiting injection site reactions occurred in 9 of 10 animals. Pre‐ and post‐treatment skin biopsies showed reduced hyperkeratosis, but increased fibrosis, with 1 of 4 alpacas remaining positive on skin scraping for mange. In conclusion, alpacas require a higher eprinomectin dose (5.0 mg/kg s.c.) than cattle, to reach comparable plasma concentrations.     

Influence of Bacillus subtilis GCB‐13‐001 on growth performance,nutrient digestibility,blood characteristics,faecal microbiota and faecal score in weanling pigs

The present study investigated the influence of Bacillus subtilis GCB‐13‐001 on growth performance, nutrient digestibility, blood characteristics, faecal microbiota and faecal score in weanling pigs. A total of 120 weaning pigs [(Landrace × Yorkshire) × Duroc; 7.73 ± 0.75 kg (28 days of age)] were randomly allotted into three treatments according to their initial body weight (BW) and gender in a 6‐week experiment. There were 8 replication pens in each treatment, with five pigs/pen. Dietary treatment groups were as follows: (a) basal diet (CON), (b) CON + 0.1% Bacillus subtilis GCB‐13‐001 1 × 10⁸ CFU/kg (T1) and (c) CON + 0.1% Bacillus subtilis GCB‐13‐001 1 × 10⁹ CFU/kg (T2). Days 1 to 7, the BW and ADG with T2 treatment were higher (p < .05) than CON treatment, as well as F:G showed trends in linear reduction (p < .1). Days 8 to 21, the BW and ADG were improved (p < .05) in pigs offered T1 and T2 diets compared with CON diet. Days 22 to 42, BW and ADG were higher (p < .05) in pigs fed T2 diet than CON and T1 diets, and the pigs fed T1 diet had higher BW than CON treatment. Overall, the ADG with the T2 treatment was higher (p < .05) than that with the T1 and CON treatments, and pigs offered T1 treatment had higher (p < .05) ADG than CON treatment. Moreover, F:G ratio were significantly decreased (p < .05) by T2 treatment compared with CON treatment. The faecal Lactobacillus counts were improved, and E. coli counts were reduced (p < .05) in pigs fed T2 diet compared with CON at the end of the experiment. In conclusion, supplementation of 0.1% Bacillus subtilis GCB‐13‐001 1 × 10⁹ CFU/kg has shown a beneficial effect in improving BW, increase ADG, decrease F:G ratio.     

Effects of chestnut tannins on intestinal morphology,barrier function,pro‐inflammatory cytokine expression,microflora and antioxidant capacity in heat‐stressed broilers

A study was conducted to evaluate the effects of chestnut tannins (CT) on intestinal morphology, barrier function, pro‐inflammatory cytokine expression, microflora and antioxidant capacity in heat‐stressed broilers. Four hundred 28‐day‐old male Ross 308 broilers were randomly assigned into four groups, with 10 replicates per group and 10 broilers per replicate. The broilers in the normal (NOR) group were kept at 22 ± 1°C and fed the basal diet, and each of the other three groups were treated with cyclic heat (33 ± 1°C from 0800 to 1800 and 22 ± 1°C from 1800 to 0800) and fed the basal diet with 0 (HT), 1 (CT1) or 2 (CT2) g of CT/kg of diet. The experiment lasted for 14 days. Compared with the HT group, broilers in the NOR and CT2 groups had higher (p < .05) average daily gain and villus height in the jejunum and lower serum d ‐lactate (p < .001) and diamine oxidase (p < .01) levels. The addition of 2 g CT/kg of diet increased the total antioxidant capacity (p < .001) and superoxide dismutase activities (p < .05) and zonula occludens‐1 mRNA expression level (p < .05) and decreased the malondialdehyde concentration (p < .01) and mRNA expression levels of interleukin‐6 (p < .001) and nuclear factor kappa B (p < .001) in the jejunal mucosa of heat‐stressed broilers. The populations of Escherichia coli and Clostridium in the jejunum (p < .01) and caecum (p < .05) of broilers in the HT group were higher than those in the NOR and CT2 groups. In conclusion, the addition of 2 g CT/kg of diet seemed to be a feasible means of alleviating the negative effects of heat stress on the growth performance and intestinal function of broilers.     

Characterization of anti‐Müllerian hormone in a case of bovine male pseudohermaphroditism

The current report aimed to characterize plasma anti‐Müllerian hormone (AMH) in bovine male pseudohermaphroditism. The blood AMH concentration in a Japanese Black male pseudohermaphrodite calf was compared with pre‐ and post‐pubertal male and female calves and castrated calves. The concentration in the case was higher than in post‐pubertal males, castrated males, and pre‐ and post‐pubertal female calves (p < .05), but similar to that in pre‐pubertal male calves. After extraction of the testes, the concentration in the case dropped to a certain extent. The extracted testes expressed AMH, as detected by immunohistochemistry. This study is the first to show the characterization of AMH in a male pseudohermaphrodite calf. AMH levels in peripheral blood might be useful to diagnose male pseudohermaphroditism in cattle.