Short‐term methionine supplementation during the early post‐partum period in primiparous rabbits improves prolificacy associated with an increase in serum concentrations of IGF‐I

The aim of this study was to evaluate the effects of methionine supplementation on energy metabolism and reproductive performance during the early post‐partum period in primiparous does. Forty nulliparous New Zealand White does were used. Females were randomized in two groups at calving: the control group (n = 20) was fed with the basal diet, and the methionine group (n = 20) was fed the basal diet plus 1 g/animal/day of methionine from the day of calving to 4 days post‐partum. Results showed that methionine supplementation increased (p = 0.032) the concentration of insulin‐like growth factor‐1 with respect to control group 4 days post‐partum. It similarly increased the prolificacy (p = 0.03), the number of kits born alive per litter (p = 0.06) and the body gain weight of the litter during supplementation (p = 0.035). These results were observed despite the does in the methionine group having a deeper negative energy balance than the does in the control group. Finally, methionine supplementation did not affect receptivity (p = 0.23), fertility (p = 0.49), the number of kits born dead per litter (p = 0.86) insulin and metabolites as glucose, non‐esterified fatty acids and triglycerides. In conclusion, our results show that methionine supplementation during the first 4 days of the post‐partum period in rabbits increases total litter size and the corporal weight of kits and is associated with an increase in blood concentration of IGF‐1.     

Characteristics of testicular cell development of 5‐day‐old mice in culture in vitro

The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5‐day‐old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5‐day‐old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO₂ atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli‐like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast‐like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia‐like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.     

Pregnancy‐Induced ISG‐15 and MX‐1 Gene Expression is Detected in the Liver of Holstein–Friesian Heifers During Late Peri‐Implantation Period

The bovine embryonic signal interferon‐τ (IFN‐τ) produced by the trophoblast is known to pass through the uterine fluid towards the endometrium and further into the maternal blood, where IFN‐τ induces specific expression of interferon‐stimulated gene expression (ISG), for example in peripheral leucocytes. In sheep, it was shown experimentally by administration of IFN‐τ that ISG is also detectable in the liver. The objective was to test whether ISG can be detected in liver biopsy specimens from Holstein–Friesian heifers during early pregnancy. Liver biopsies were taken on day 18 from pregnant and non‐pregnant heifers (n = 19), and the interferon‐stimulated protein 15 kDa (ISG‐15) and myxovirus‐resistance protein‐1 (MX‐1) gene expression was detected. The expression of both MX‐1 (p: 24.33 ± 7.40 vs np: 9.00 ± 4.02) and ISG‐15 (p: 43.73 ± 23.22 vs 7.83 ± 3.63) was higher in pregnant compared to non‐pregnant heifers (p < 0.05). In conclusion, pregnancy induced ISG‐15 and MX‐1 gene expression in the liver already at day 18 in cattle.     

Reproductive response of fat‐tailed Barbarine ewes subjected to short‐term nutritional treatments including spineless cactus (Opuntia ficus‐indica f. inermis) cladodes

Reproductive outputs in fat‐tailed Barbarine sheep in central Tunisia are often low because of feed shortage and the low nutritive value of diets. Supplementation with conventional concentrates is economically unsuitable in central Tunisia, so more cost‐effective and sustainable alternative feeding strategies need to be developed. We tested effects of short‐term nutritional treatments including cactus cladodes during the induction of ‘male effect’ on fertility and prolificacy parameters (follicular growth, ovulatory response and early embryo losses). One hundred and twenty ewes were distributed in 4 equal groups balanced for live weight grazed natural pastures and were supplemented for 21 days, starting day 10 after introduction of rams, with cactus cladodes (CA), cactus cladodes and soybean meal (CAS), concentrate (CC) or only soybean meal (S). Nutritional treatment did not affect live weight in this experiment. Ewes receiving cactus had higher number of large pre‐ovulatory follicles (≥6 mm; 1.08 ± 0.05), between days 14 and 19 after introduction of rams, than females in the CC and S ewes (0.64 ± 0.06; p < 0.05). However, there were no differences in the onset of oestrous behaviour in response to ‘male effect’ or in the number of corpora lutea. Average ovulation rates were 1.42 ± 0.16 for CC, 1.47 ± 0.13 for CAS, 1.31 ± 0.15 for CA and 1.31 ± 0.13 for S groups respectively. Finally, reproductive wastages at day 35 after mating were not different between groups being 0.33 ± 0.19 for CC, 0.60 ± 0.17 for CAS, 0.43 ± 0.16 for CA and 0.31 ± 0.15 for S groups respectively. It is concluded that Barbarine ewes fed nutritional treatments including cactus performed similarly to those receiving diets including conventional concentrate feeds.     

Effects of high‐fat and high‐carbohydrate diets on fat and carbohydrate oxidation and plasma metabolites in healthy cats

High‐fat (HF) or high‐carbohydrate (HC) diets (30% fat, 18.9% carbohydrate; HF and 10% fat, 46.3% carbohydrate; HC) and lengths of adaptation were investigated in cats (Felis catus; 10 ± 2 months, 3.6 ± 0.3 kg). Cats randomly received each treatment for 14 days in a crossover design with a 14‐day washout period between each diet. Three 22‐h indirect calorimetry studies were conducted after acute (day 0), semichronic (day 4) and chronic (day 13) dietary exposure. Blood samples were collected after a 24‐h fast on days 1, 5 and 14. When cats consumed the HC and HF diet, oxidation of the restricted nutrient exceeded intake while oxidation of the nutrient in excess matched intake. Mean max energy expenditure (EE) of cats consuming the HF and HC diet were 107 and 102 kcal/kg^0.67/day and occurred at a mean of 4 and 12 h post‐feeding respectively. Maximal fat (0.90 g/h) and carbohydrate (carbohydrate; 1.42 g/h) oxidation were attained at 26 min and 10.4 h post‐feeding respectively. The changes observed in macronutrient oxidation and EE suggest that cats adapt whole‐body nutrient metabolism in response to changes in dietary macronutrient content, but may require longer than 14 day to adapt to a macronutrient that is present at a lower concentration in the diet.     

Safety of standardized Macleaya cordata extract in an eighty‐four‐day dietary study in dairy cows

The objective of this study was to evaluate the safety of a standardized Macleaya cordata Extract Product (MCEP) containing the quaternary benzophenanthridine alkaloids, sanguinarine and chelerythrine, when fed to dairy cows. Thirty‐six dairy cows were randomized into three groups with twelve cows/treatment in two replica pens for each treatment group: control (C) without MCEP added to feed, treatment 1 (SANG‐1000) with MCEP added to feed at 1,000 mg/animal/day (1.5 mg/kg bw/day) and treatment 2 (SANG‐10000) with MCEP added to feed at 10,000 mg/animal/day (15.5 mg MCEP/kg bw/day). After two weeks of acclimation, animals were observed for an 84‐day experimental period, with body weight, feed intake and milk production measured daily. Milk composition was analysed every two weeks. Haematological analyses were performed on Day 0 and Day 84, and clinical chemistry analyses were performed on Day 84 of the study. There was no statistically significant difference (p > .10) among the three groups on body condition score, milk production or milk composition over the study period. There were no significant differences in body weight gain or feed consumption among the three groups. Animals in the SANG‐10000 group had significantly higher mean corpuscular volume (MCV) than the C group (p < .1) and lower mean corpuscular haemoglobin concentration (MCHC) than the SANG‐1000 group (p < .1). Concentrations of sanguinarine and chelerythrine in milk samples collected on Day 84 were below the detection limit (LOD) as measured by high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS/MS). In conclusion, this study presents compelling data supporting the hypothesis that the test product MCEP, when included in the TMR at up to 10,000 mg/animal/day (15.5 mg MCEP/kg bw/day), is well tolerated by dairy cows.     

Effects of the presence of grazing‐experienced heifers on the development of foraging behavior at the feeding station scale for first‐grazing season calves

In this study, effects of grazing‐experienced heifer presence on foraging behavior development at the feeding station (FS) scale for first‐grazing season calves were determined. A group of four calves grazing alone (C), and another comprising four calves (Wc) and three grazing‐experienced heifers (Wh), were alternately stocked every day for 2 h on the same pasture for 26 days. The foraging time budget for Wc was significantly longer than that for C (P < 0.05) on day 7, and was similar to that for Wh on all days. Bite rate per FS was significantly higher for Wc (15.5 bite/min) than for C (13.2 bite/min) from day 1 to 14 (33.4 vs. 29.0 bite/min, respectively; P < 0.01) and significantly lower than that for Wh on all days. The number of steps taken between adjacent FSs by Wc (4.7) was significantly lower than that for C (7.2) on days 1 and 14 (2.1 vs. 2.9 steps, respectively; P < 0.01) and was similar to Wh on all days. This suggests that grazing‐experienced heifers have positive effects on the foraging behavior development of new‐season grazing calves through 14 days after the start of stocking.     

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.     

Effects of Dietary Supplementation of β‐hydroxy‐β‐methylbutyrate on Sow Performance and mRNA Expression of Myogenic Markers in Skeletal Muscle of Neonatal Piglets

The effects of dietary β‐hydroxy‐β‐methylbutyrate (HMB) supplementation during gestation on reproductive performance of sows and the mRNA expression of myogenic markers in skeletal muscle of neonatal pigs were determined. At day 35 of gestation, a total of 20 sows (Landrace × Yorkshire, at third parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 4 g/day β‐hydroxy‐β‐methylbutyrate calcium (HMB‐Ca) until parturition. At parturition, the total and live litter size were not markedly different between treatments, however, the sows fed HMB diet had a decreased rate of stillborn piglets compared with the sows fed the control (CON) diets (p < 0.05). In addition, piglets from the sows fed HMB diet tended to have an increased birth weight (p = 0.08), and a reduced rate of low birth weight piglets (p = 0.05) compared with piglets from the CON sows. Nevertheless, lower feed intake during lactation was observed in the sows fed the HMB diet compared with those on the CON diet (p < 0.01). The relative weights of the longissimus dorsi (LD) and semitendinosus (ST) muscle were higher (p < 0.05) in neonatal pigs from the HMB than the CON sows. Furthermore, maternal HMB treatment increased the mRNA levels of the myogenic genes, including muscle regulatory factor‐4 (MRF4, p < 0.05), myogenic differentiation factor (MyoD) and insulin‐like growth factor‐1 (IGF‐1, p < 0.01). In conclusion, dietary HMB supplementation to sows at 4 g/day from day 35 of gestation to term significantly improves pregnancy outcomes and increases the expression of myogenic genes in skeletal muscle of neonatal piglets, but reduces feed intake of sows during lactation.     

Response of lactating dairy cows to degree of steam‐flaked barley grain in low‐forage diets

This study was conducted to investigate the effects of processing method (grinding vs. steam flaking) and increasing densities of steam‐flaked barley grain on dry matter intake (DMI), rumen pH and fermentation characteristics, digestibility of dry matter in the total digestive tract (DDTT), and milk production of dairy cows. Eight multiparous mid‐lactation Holstein cows averaging 103 ± 24 DIM, 44.5 ± 4.7 kg milk/day and weighing 611 ± 43 kg at the start of the experiment were used in a replicated 4 × 4 Latin square design with 21‐day periods. Cows were fed diets consisting of (DM basis) 23.8% corn silage, 13.5% chopped alfalfa hay and 62.7% concentrate. The dietary treatments were either ground barley (GB) using a hammer mill or steam‐flaked barley (SFB) – varying density at 390, 340 or 290 g/l. Processing method (GB vs. SFB) did not affect DMI (23.6 kg/day on average), DDTT (71.0% on average), milk yield (43.4 kg/day on average), milk components, rumen pH and molar proportions of acetate, propionate, butyrate and sorting activity. Ruminal isovalerate concentration tended (p = 0.06) to be higher for cows fed GB than those fed SFB‐based diets. Decreasing the density of SFB from 390, 340 to 290 g/l tended to linearly increase DMI (p = 0.09), decrease total solids percentage of milk (p = 0.10) and linearly decreased milk urea nitrogen (12.8, 12.4 and 12.1 mg/dl; p = 0.04); also, the sorting index (SI) of the particles retained on the 19.0‐mm sieve without affecting the SI of the particles retained on 8.0‐mm, 1.18‐mm or passed through 1.18‐mm sieve (p = 0.05). These results indicated the limited effects of processing method (grinding vs. steam flaking) and densities of SFB (390, 290 or 290 g/l) on cows’ performance and feed utilization for dairy cows fed low‐forage diets. Therefore, both processing methods could be recommended under current feeding conditions of dairy cows.     

Effect of short‐term supplementation with rumen‐protected fat during the late luteal phase on reproduction and metabolism of ewes

This study was designed to study the effect of short‐term supplementation with rumen‐protected fat during the late luteal phase on reproduction and metabolism of sheep during breeding season. Seventy‐six ewes (Rahmani, Barki and Awassi × Barki) were allocated to two groups considering genotype: the control ewes (C‐group) received a maintenance diet, and the fat‐supplemented ewes (F‐group) received the maintenance diet plus 50 g/head/day of rumen‐ protected fat (Megalac) for 9 days during which oestrus was synchronized. The latter had been accomplished using double intramuscular injection of prostaglandin F_2α (PGF_2α) 11 days apart. Ovarian activity, serum concentration of cholesterol, glucose, insulin and reproductive performance variables were recorded. Data were analysed considering treatment (group) and genotype. Supplementation had positive effects on the overall mean serum concentrations of cholesterol (p < 0.05), glucose (p < 0.05) on day 6 of nutritional treatment and insulin (p = 0.07) on day 8. Fat supplementation did not affect the total number of follicles, follicle populations and ovulation rate. However, fat‐supplemented Rahmani ewes tended to have higher ovulation rate compared with other breeds (treatment × breed interaction, p = 0.06). Treatment also did not affect the mean concentration of serum estradiol or progesterone. Supplemented ewes had higher conception (p = 0.06) and lambing rates (p < 0.05) compared with control. In conclusion, short‐term supplementation with rumen‐protected fat as a source of energy around breeding time improved metabolism, conception and lambing rates of ewes without effects on steroidogenic capacity and ovarian activity being apparent.     

Metabolomic profiling in umbilical venous plasma reveals effects of dietary rumen‐protected arginine or N‐carbamylglutamate supplementation in nutrient‐restricted Hu sheep during pregnancy

Maternal nutrient restriction during pregnancy is a major problem worldwide for human and animal production. Arginine (Arg) is critical to health, growth and reproduction. N‐carbamylglutamate (NCG), a key enzyme in arginine synthesis, is not extensively degraded in rumen. The aim of this study was to investigate ameliorating effects of rumen‐protected arginine (RP‐Arg) and NCG supplementation on dietary in undernourished Hu sheep during gestation. From day 35 to 110 of gestation, 32 Hu ewes carrying twin foetuses were randomly divided into four groups: a control (CG) group (n = 8; fed 100% National Research Council (NRC) requirements for pregnant sheep), a nutrient‐restricted (RG) group (n = 8; fed 50% NRC requirements, which included 50% mineral–vitamin mixture) and two treatment (Arg and NCG) groups (n = 8; fed 50% NRC requirements supplemented with 20 g/day RP‐Arg or 5 g/day NCG, which included 50% mineral–vitamin mixture). The umbilical venous plasma samples of foetus were tested by ¹H‐nuclear magnetic resonance. Thirty‐two differential metabolites were identified, indicating altered metabolic pathways of amino acid, carbohydrate and energy, lipids and oxidative stress metabolism among the four groups. Our results demonstrate that the beneficial effect of dietary RP‐Arg and NCG supplementation on mammalian reproduction is associated with complex metabolic networks.     

Effect of n‐3 PUFA‐rich fish oil supplementation during late gestation on kidding,uterine involution and resumption of follicular activity in goat

We have shown that dietary supplementation of n‐3 polyunsaturated fatty acid (n‐3 PUFA)‐rich fish oil (FO) around the breeding time improved the utero‐ovarian functions in the goat. Here, we investigated the effect of FO supplementation during the periparturient period on serum n‐3 PUFA, prostaglandin F_2α metabolite (PGFM), placental expulsion, uterine involution, resumption of oestrus and neonatal vigour. Rohilkhandi goat in advanced gestation (n = 16) was divided into two equal groups. One group was supplemented with FO containing 26% n‐3 long‐chain PUFA at the rate of 156 mg per kg body weight, while the control group was fed isocaloric palm oil (PO) from ?3 to +3 week of kidding. Dietary FO increased serum concentration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by 7.3‐ and 6.6‐fold, respectively, after 6 weeks of supplementation. Goats in FO group expelled the foetal membranes 99.1 min earlier (p < .01) than those of PO group. Further, dietary FO significantly decreased the serum PGFM on day 7 post‐partum. However, no difference was found on uterine involution, which was complete by day 20 post‐partum in either group. Resumption of follicular activity by day 5 post‐partum was 87.5% in the FO as compared to 25% in the PO group (p < .05). Similarly, occurrence of behavioural oestrus by day 90 post‐partum was 57.1% in goats of the FO group while none of does was in the PO group (p < .01) expressed oestrus. It was concluded that feeding FO‐rich diet during ?3 to +3 weeks of kidding decreased the PGFM till day 7 post‐partum, hastened the expulsion of foetal membranes and reduced the time from kidding to first post‐partum oestrus in Rohilkhandi does.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.     

2‐Hydroxy‐4‐methylthio butanoic acid and DL‐methionine for Japanese quails in production

An experiment was performed using 1,000 laying Japanese quails to assess the availability of two alternative dietary methionine sources. Treatment 01 = Basal Feed that is deficient in digestible methionine + cystine (Met + Cys). The other treatments were constituted by Met + Cys levels of 0.8, 1.60 and 2.40 g/kg, supplemented with DL‐Methionine‐99%, HMTBA‐88% and HMTBA‐84%, being 10 treatments in total. The following characteristics were studied: feed intake (g/bird/day), egg production (egg/day × 100), egg weight (g/egg), egg mass (g/egg), feed conversion per egg dozen (kg feed/dozen eggs), feed conversion per egg mass (kg feed/kg eggs), relative yolk weight (g/100 g of egg), relative albumen weight (g/100 g of egg), relative shell weight (g/100 g of egg), shell thickness (mm) and specific gravity (g/cm³). In general result comment, supplemental methionine sources must be included in the poultry diet. The different methionine sources affect the performance of quails, and the increase in the levels within each source improves the performance variables. Significant effect was observable on performance variables and egg quality variables, being that DLM‐99% is superior to the other sources. The HMTBA‐88% source is superior to the HMTBA‐84% source for the same aforementioned variables. In conclusion, the bioefficacy values of the HMTBA‐88% and HMTBA‐84% sources compared to the DLM‐99% source on an equimolar basis were 81 and 79%, respectively, for the performance variables, and 83 and 74 while the methionine sources were equivalent for the variables related to egg quality.     

Reproductive performance of breeder quails fed diets supplemented with L‐cysteine‐coated iron oxide nanoparticles

The objective of this study was to investigate the effects of L‐cysteine‐coated iron oxide nanoparticles on reproductive performance in breeder quails. The five treatment diets consisted of (i) negative control diet not supplemented with iron, (ii) positive control diet supplemented with 60 mg/kg of Fe₃O₄ and (iii) experimental diets supplemented with 0.6, 6 and 60 mg/kg of L‐cysteine‐coated iron oxide nanoparticles. A total of 100 seven‐day‐old quail chicks were weighed and randomly placed to five groups of five replicate cages. Four quails (one male and three females) were raised in each cage (50 × 15 × 17 cm). Egg production, feed consumption and egg weight were recorded daily and calculated on a hen per day basis. Egg components, fertility, hatchability and day‐old chicks hatched from their eggs were measured at the end of the experiment. The percentage of egg production and egg mass of the 6 mg/kg Fe₃O₄‐Cys NPs group were significantly higher than those of the control groups. Throughout the experimental period, the highest weekly egg weight was recorded for the 60 mg/kg Fe₃O₄‐Cys NPs group. Fertility was improved by diet supplemented with iron, both FeSO₄ and Fe₃O₄‐Cys NPs. The breeder fed Fe₃O₄‐Cys NPs had the highest day‐old chicks weight. The results of this study showed that Fe₃O₄ nanoparticles that were coated by L‐cysteine could improve availability and utilization of iron in diet. Finally, it was proposed that Fe₃O₄‐Cys NPs could be used as feed additives in quails.     

Differential gene expression of Eph‐ephrin A1 and LEPR‐LEP with high or low number of embryos in pigs during implantation

The objective of this study was to ascertain whether mRNA and protein expressions of implantation‐related genes (erythropoietin‐producing hepatocellular receptor–ligand A1, Eph‐ephrin A1 and leptin receptor–leptin, LEPR‐LEP) differed between pigs with high and low number of embryos, and whether these differences in gene expression might affect embryo implantation. Experimental pig groups (n = 24) for high and low number of embryos were prepared by altering the number of eggs ovulated in pre‐pubertal gilts treated with 1.5 × (High) or 1.0 × (Low) PG600 ([400 IU PMSG + 200 IU hCG]/dose, AKZO‐NOBEL). Gilts expressing oestrus were artificially inseminated twice and maintained in breeding and gestation until the reproductive tract was collected on day 22 of pregnancy. At slaughter, the reproductive tracts from each pregnant gilt from each treatment were immediately processed to collect samples for RNA and protein analysis. Within each gilt, three conceptus points were sampled, one from each horn and then a random conceptus within the tract. At each conceptus point, endometrial attachment site, chorion–allantois and embryo were collected and immediately frozen in liquid nitrogen. Number of corpus luteum (CL) (35.4 vs. 12.6) and total embryo number (18.8 vs. 10.2) were greater in the high‐embryo compared to the low‐embryo group, respectively (p < .05). Real‐time qPCR results showed that Eph‐ephrin A1 mRNA expression was less in the high‐embryo (p < .05) compared to the low‐embryo group. In addition, Western blotting analysis indicated that Eph‐ephrin A1 and LEP protein expression at endometrial attachment site in high‐embryo was less (p < .05) compared to low‐embryo group. It was also noted that mRNA expression of Eph‐ephrin A1 and LEPR‐LEP was greater in pregnant than non‐pregnant gilts (p < .05). Moreover, mRNA expression of Eph‐ephrin A1 (p < .05) and LEPR‐LEP was greatest at endometrial attachment site among all three tissues. There was a positive correlation between expressions of Eph‐ephrin A1, LEPR‐LEP and embryo length with the correlation coefficient 0.31–0.59. For Eph‐ephrin A1, the highest correlation coefficient appeared between Eph A1 expression and normal embryo number, between ephrin A1 expression and embryo length. For LEPR‐LEP, the highest correlation coefficient appeared between LEPR‐LEP expression and ovary weight (0.79 for both, p < .05), followed by embryo length and weight. The results of this study suggest that low expression of Eph‐ephrin A1 and LEPR‐LEP is somehow related to increased embryo number during implantation and that endometrial attachment site might be the main target tissue of these gene products. Yet, the increased expression of Eph‐ephrin A1 and LEPR‐LEP appeared associated with increased embryo growth (length and weight) and ovary weight, Eph‐ephrin A1 and LEPR‐LEP might play roles in the regulation of embryo implantation in pigs.     

A rapid analytical method of major milk proteins by reversed‐phase high‐performance liquid chromatography

A reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method with rapid and automated analysis, good separation, high resolution, high accuracy and reproducible results was successfully developed and used to separate and quantify the major cow milk proteins within 30 min analytical time. Standard solutions of single purified cow milk proteins were used to develop calibration equations. The RP‐HPLC method was validated with respect to intra‐day repeatability, inter‐day precision, linearity, accuracy and limit of detection (LOD). The recoveries of the RP‐HPLC analyses of major milk proteins from cows ranged 71.0–114%, the inter‐day precision was expressed as the relative standard deviation, and the ranged from 1.51 to 4.60% and the LOD ranged from 0.08 g/L to 0.28 g/L. Major proteins in cow were quantified according to the chromatographic profiles. Results showed that a rapid RP‐HPLC method for quantifying the major cow milk proteins was developed, which could be used to determine milk protein contents in the dairy industry.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

Acute and Chronic Effects of a Contraceptive Compound RTI‐4587‐073(l) on Testicular Histology and Endocrine Function in Miniature Horse Stallions

The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI‐4587‐073(l). Six miniature stallions were used in this experiment. The treatment group (n = 3) received one oral dose of 12.5 mg/kg of RTI‐4587‐073(l), and the control group (n = 3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5 weeks before treatment and for 6 weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1 day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6 weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p < 0.05), while the number of immature germ cells increased in ejaculates from treated animals (p < 0.05). Serum concentrations of inhibin and follicle‐stimulating hormone did not change. Testosterone concentrations initially transiently decreased (p < 0.05) after administration of RTI‐4587‐073(l), and increased several days later (p < 0.05). Testicular content of testosterone and estradiol 17‐β was lower in treated stallions than in control stallions on Day 1 after treatment (p < 0.05). Severe disorganization of the seminiferous tubules, significant loss of immature germ cells and complete depletion of elongated spermatids were observed in testicular biopsies obtained from treated stallions 1 day, 2 and 4 weeks after treatment. These changes were still present in the testicular samples taken from treated stallions after castration. The results of this study confirmed that RTI‐4587‐073(l) has antispermatogenic effects in stallions. Furthermore, we concluded that this compound causes acute sloughing of immature germ cells from the seminiferous tubules. RTI‐4587‐073(l) has significant but transient effects on Leydig cell function in stallions.