Undernutrition During Foetal and Post‐Natal Life Affects Testicular Structure and Reduces the Number of Sertoli Cells in the Adult Rat

To test whether undernutrition during foetal to pre‐pubertal life would have long lasting effects on testicular histology in adult male offspring, eleven adult Sprague–Dawley pregnant rats were divided into two groups: Control group, n = 4, fed ad libitum, during gestation and lactation (until 25 day post‐partum). Underfed group pregnant females (n = 7) were kept in cages where only dams had access to food (standard rat chow, 33.5% of ad libitum intake of Control group pregnant dams). After parturition, litters were adjusted to either 14 (Underfed group) or eight (Control group) pups. Mothers were weighed weekly. At 25 day of age pups were weaned, housed at four animals per cage, fed ad libitum and weighed weekly until euthanized at 100 day of age. Testes were processed for standard histology and morphometrical evaluation. At weaning, mother weight was lower in Underfedthan in Control group (mean ± SD): 214.1 ± 26.2 g vs 361.9 ± 33.1 g. Body weight at 100 days of age (254 ± 26.9 g vs 342.4 ± 10.2 g, p ≤ 0.001), testicular weight (1.29 ± 0.16 g vs 1.45 ± 0.13 g, p = 0.03), number of Sertoli cells per seminiferous tubule cross section (18.2 ± 1.2 vs 20.2 ± 1.3, p ≤ 0.01) and per testis (30.5 ± 4.2×10⁶ vs 36.0 ± 5.4×10⁶) were lower (p < 0.05) in Underfed than in Control group. This is the first report stating that foetal to pubertal subnutrition is accompanied by changes in testicular structure and lower Sertoli cell numbers in adult life, strongly suggesting lower daily sperm production.     

The Expression of Epidermal Growth Factor (EGF) and its Receptor (EGFR) During Post‐Natal Testes Development in the Yak

Growth factors play critical role in cell proliferation, regulate tissue differentiation and modulate organogenesis. Several growth factors have been identified in the testes of various mammalian species in last few years. In present investigation, the objective was to determine the expression of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in yak testicular tissue by relative quantitative real time polymerase chain reaction (RT‐PCR), Western blot (WB) and immunohistochemistry (IHC) from mRNA and protein levels. The testicular tissues were collected from male yak at 6 and 24 months old. Results of RT‐PCR and WB showed that the expression quantity of EGF and EGFR at 24 months of age was higher than at 6 months, and the increase rate of EGFR on mRNA and protein levels was higher than the increase rate EGF during post‐natal testes development. Positive staining for EGF and EGFR was very low and mainly localized to Leydig cells testes at 6 months of age with immunohistochemistry, and seminiferous tubules were not observed. At 24 month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. However, EGF and EGFR were localized to preferential adluminal compartment and basal compartment in the seminiferous tubules, respectively. In conclusion, the findings in present studies suggest that EGF and EGFR as important paracrine and/or autocrine regulators in yak testes development and spermatogenesis.     

Pre‐ and Post‐Partum Mild Underfeeding Influences Gene Expression in the Reproductive Tract of Cyclic Dairy Cows

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post‐partum. This study investigated the effects of pre‐ and post‐partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post‐partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post‐partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi‐ and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post‐partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post‐partum patterns of IGF‐1, insulin, β‐hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid‐related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post‐partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post‐partum dairy cows.     

Prevalence and Pathogenicity of Cryptosporidium suis in Pre‐ and Post‐weaned Pigs

A total of 4338 faecal samples, 135 of sows, 3368 of pre‐weaned and 835 of post‐weaned piglets from eight farms in South Bohemia, Czech Republic were collected and examined for Cryptosporidium infection. No sow, but 5.7% pre‐weaned and 24.1% post‐weaned piglets were positive for Cryptosporidium infection. No relationship was found between diarrhoea and Cryptosporidium infection in any of the different age groups (pre‐ and post‐weaned piglets). Four piglets, which were sporadically shedding cryptosporidia in faeces, were necropsied. Neither clinical signs of diarrhoea nor macroscopical changes were found. Histologically, a moderate infection of cryptosporidia was detected in the glandular epithelium along the large intestine, with predisposition to the ansa centralis of the colon. No inflammatory response in the lamina propria was observed. Cryptosporidia were also commonly found in the glandular epithelium of submucosal lymphoglandular complexes in the colon. Cryptosporidium isolates from all farms were identified as Cryptosporidium suis using molecular markers (SSU rRNA). All of the C. suis strains obtained were larger [6.2 (6.0–6.8) × 5.5 (5.3–5.7) μm] than any isolate described so far [4.6 (4.4–4.9) × 4.2 (4.0–4.3) μm] and did not appear to be infective for neonatal BALB/c mice.     

The Distribution of Gastrin,Somatostatin and Glucagon Immunoreactive (IR) Cells in Ostrich Stomach During the Pre‐ and Post‐hatching Period

Peptides of the gastrointestinal tract play a significant role in the digestive processes and the development of the body; therefore, it is important to have an understanding of location and distribution of gastrin, somatostatin and glucagon immunoreactive (IR) cells in the stomach mucosa of growing birds. For this purpose, 6 embryos and 37 chicks from an ostrich farm in Latvia were used. Tissue samples were collected from the proventriculus – superficial and deep glandular region and from the ventriculus – side wall and pyloric region. The number of cells was determined in 10 mucosal fields of each tissue sample. For statistical analysis, the one‐way anova method was used. Gastrin IR cells regarding the stomach mucosa were found only in the pyloric region. Somatostatin IR cells were most densely located in the pyloric region too, but some cells were also discovered in the mucosa of proventriculus and ventriculus. Glucagon IR cells were found in the epithelium of the deep glands of the proventriculus and only some cells of the superficial glands of the proventriculus, and the ventriculus side wall mucosa. Gastrin and somatostatin IR cells were present in a comparatively large quantity in the ostrich chicks' ventriculus – pyloric region yet not long before hatching. They were located deep in the mucosa of pyloric glands, and their number tended to increase with birds advancing in age.     

Structural Changes of Goat Parotid Salivary Gland: Pre‐ and Post‐Weaning Periods

Previously, the structure of the adult goat parotid salivary glands (PGs) was studied. However, little information was elucidated of the juvenile ones. This study aimed to clarify the correlations between the structure of goats' PGs and the nature of food intake among milk‐suckling kids (MSKs) and diet‐fed goats (DFGs). The secretory endpieces of the goats' PGs are of the pure serous type. The serous cells in MSKs showed apical accumulation of numerous secretory granules (SGs) of smaller size and of more intense positive periodic acid‐Schiff reaction. Ultrastructurally, most of the SGs in the DFGs contained peripherally located inclusions that showed dense reaction products for acid phosphatase. In MSKs, the PGs showed less‐developed basal infoldings, sparseness of the inter‐cellular inter‐digitations, fewer inter‐cellular canaliculi and microvilli and also less‐developed myoepithelial cells with fewer and shorter cytoplasmic processes. In conclusion, the less‐developed membrane specializations and myoepithelial cells, as well as the accumulated SGs in the PGs of MSKs, suggest that it secretes less saliva with a little secretory activity than that of DFGs, which may be correlated with the reduced masticatory activity.     

Distribution and Characterisation of Goblet Cells in the Large Intestine of Ostriches during the Pre‐ and Post‐Hatch Period

The role of goblet cell secretion, containing mucopolysaccharides, in the formation of a protective barrier of intestinal mucosa and transportation of the intestinal content has been described quite extensively. However, information on the quality composition of mucopolysaccharides and its changes in the intestinal tract of ostrich chicks, especially in the large intestinal segments, is unavailable. In the current study, ostrich embryos/chicks (n = 6/36) of both sexes were used shortly before hatching and during the first months of the post‐hatch period. Tissues for histology were taken from the large intestine: the medium segments of the caecum, proximal and distal parts of colon. By using histochemical reactions, the differentiation of goblet cells as well as chemical composition of mucopolysaccharides was carried out. The cells contained acid (AB+), neutral (PAS+) and mixed (AB/PAS+) mucopolysaccharides. The number of goblet cells in the large intestine per unit area of mucosa increased towards the cloaca, and it was the highest in the distal part of the colon. The qualitative goblet cell composition in different large intestinal parts was different in all ages. In the caecum, goblet cells containing acid and mixed mucopolysaccharides dominate post‐hatch, whereas in the colon, goblet cells containing acid mucopolysaccharides predominated. The most rapid changes in the qualitative goblet cell composition occur during the first week post‐hatch when in all the intestinal segments the proportion of cells containing acid mucopolysaccharides continuously increased.     

Effects of Pre‐Weaning Energy Substitutions on Post‐Weaning Follicle Development,Steroid Hormones and Subsequent Litter Size in Primiparous Sows

The present study investigated the effects of pre‐weaning energy substitutions on follicular development, endocrine characteristics and subsequent litter size in primiparous sows. Sows were fed a standard lactation diet (14.1 DE MJ/kg) and then allocated to a Control (C, n = 24), Fat (F, n = 23), Sugar (S, n = 23) or post‐weaning Regumate (positive control; R, n = 22) treatment at 9 days before weaning of the C, F and S treatments. During the treatment period (8 days), 1 kg of the lactation diet was substituted with 1 kg of a fat‐rich (F, 23.85 DE MJ/kg) or sugar‐rich (S, 15.75 DE MJ/kg) substitution for F and S sows, respectively. For the R treatment, sows were weaned 8 days earlier than other treatments and fed a lactation diet at 3.5 kg with two doses of altrenogest as topdressing from 1 day before weaning until the day on which the other sows were weaned. The F treatment aimed to increase energy intake, and the S treatment aimed to elevate post‐prandial glucose and insulin concentrations. Weaning‐to‐ovulation interval tended to be reduced in the S treatment compared with C (p = 0.06) and F (p = 0.08) treatments. Body weight (BW) loss during the treatment period, post‐weaning follicle development, plasma oestradiol and pre‐weaning leptin did not differ among C, F and S sows, although BW loss was lower and leptin was higher in the R treatment. Post‐ovulatory progesterone concentration in the S treatment was higher (p < 0.05). Sows in the S and R treatments had a greater proportion of litters with larger litter sizes (p < 0.05). The outcome suggests that increasing circulating insulin and glucose concentrations during late lactation or a week of metabolic recovery positively improves subsequent litter size in primiparous sows.     

Optimizing Electrical Activation of Porcine Oocytes by Adjusting Pre‐ and Post‐Activation Mannitol Exposure Times

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre‐ and post‐activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre‐ or post‐electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3 min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2 min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3 min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1 min pre‐ and 3 min post‐activation showed significantly higher blastocyst development than 0 min pre‐ and 0 min post‐activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0 min pre‐ and 0 min post‐activation. In conclusion, exposure of oocytes to mannitol for 1 min pre‐ and 3 min post‐activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1 min pre‐ and 3 min post‐activation.     

Immunohistochemical Localization of Fibroblast Growth Factor‐2 in the Sheep Ovary and its Effects on Pre‐antral Follicle Apoptosis and Development In Vitro

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM⁺) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.     

Fine Structures of Embryonic Discs of In Vivo Post‐hatching Porcine Blastocysts at the Pre‐Primitive Streak Stage

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre‐primitive streak area of embryonic discs. The cells were 1–2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc.     

Post‐natal Changes in Testicular Concentrations of Interleukin‐1 Alpha and Beta and Interleukin‐6 during Sexual Maturation in Bulls

Based on observations in laboratory animals interleukins could be regulators of testicular development. The objects of this study were to see if interleukins (IL‐1 and IL‐6) are present in the developing bull testis and to establish the temporal patterns of concentrations of IL‐1 and IL‐6 in the bovine testis during development. Separate groups of six bull calves were castrated every 4 weeks from 5 to 33 weeks of age, and at 56 weeks of age. Mean testicular IL‐1 alpha concentrations decreased (p < 0.01) from 5 to 9 weeks of age and 13 to 21 weeks of age. Mean testicular IL‐1 beta concentrations decreased (p < 0.01) from 13 to 17 weeks of age and from 29 to 33 weeks of age. Mean IL‐1 bioactivity increased from 13 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks, decreased to 29 weeks and decreased from 33 to 56 weeks of age (p < 0.05). Mean testicular IL‐6 concentrations decreased (p < 0.05) from 9 to 13 weeks of age, increased (p < 0.05) to 21 weeks, decreased (p < 0.05) to 25 weeks, increased (p < 0.05) to 29 weeks and decreased (p < 0.01) to 56 weeks of age. In conclusion, testicular IL‐1 alpha, IL‐1 beta and IL‐6 were found in the bovine testis and concentrations were age dependent. Testicular IL‐1 alpha and IL‐1 beta concentrations were highest in the early post‐natal period; however, IL‐1 bioactivity and IL‐6 concentrations were greatest in the immediate pre‐pubertal period. These findings suggest a functional role for interleukins in testicular development in the bull.     

Impact of Food Restriction on Ovarian Development,RFamide‐Related Peptide‐3 and the Hypothalamic–Pituitary–Ovarian Axis in Pre‐Pubertal Ewes

RFamide‐related peptide‐3 (RFRP‐3), the mammalian ortholog of gonadotropin‐inhibiting hormone, has been implicated as a mediator between reproduction and energy balance. This study aimed to investigate the physiological effects of RFRP‐3 on the process of ovarian development in food‐restricted pre‐pubertal ewes. The results showed that food restriction significantly inhibited the ovarian development and follicular growth. The data of qPCR in the hypothalamic–pituitary–ovarian (HPO) axis showed that food restriction not only upregulated RFRP‐3 mRNA expression but also downregulated the mRNA expression of gonadotropin‐releasing‐hormone receptor, follicle‐stimulating hormone receptor and luteinizing hormone receptor (LHR). Immunohistochemistry of RFRP‐3 in the ovaries suggested that RFRP‐3 may regulate the follicular development. These results suggested that the changes of RFRP‐3 in response to food restriction might influence the HPO axis and inhibit ovarian development.     

Effect of Semen Collection Methods on the Quality of Pre‐ and Post‐thawed Bali Cattle (Bos javanicus) Spermatozoa

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post‐thawed semen quality. The collection methods employed were electro‐ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM + EE). A total of 25 untrained Bali bulls (age between 2 and 4 years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer‐assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM + EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM + EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2–3 and >3–4 years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM + EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.     

Expression of hLAMP‐1‐Positive Particles During Early Heart Development in the Chick

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin‐associated matrix protein‐1 (hLAMP‐1) is a matrix protein expressed within Hensen's node at Hamburger–Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5–6 and enhanced within the left pre‐cardiac field at HH stage 7. At HH stages 15–16, hLAMP‐1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP‐1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8–14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8–14 with antibodies specific to hLAMP‐1. Our results showed prominent expression of hLAMP‐1‐positive particles in the extracellular matrix associated with the pre‐cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8–9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP‐1‐stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP‐1 may be involved in the formation of the endocardial tube.     

Calcitonin Promotes Mouse Pre‐implantation Development: Involvement of Calcium Mobilization and P38 Mitogen‐Activated Protein Kinase Activation

The study was designed to examine the effects of calcitonin (CT) on the development of murine pre‐implantation embryos and possible molecular mechanisms involved in the process. In the present study, the 2‐cell embryos were treated with different concentration of CT in vitro for the indicated time and the results demonstrated that CT promoted the development of the pre‐implantation embryos in a dosage‐dependent manner by increasing the intracellular Ca²⁺ level. Furthermore, the present study showed that CT significantly increased the expression of phospho‐P38MAPK (Mitogen‐Activated Protein Kinase) of the pre‐implantation embryos by Western blots and pre‐treatment of specific P38MAPK inhibitor significantly reduced the promotion effects of CT on the embryonic development in vitro culture. Moreover, the results of intrauterine horn injection showed that the average number of embryos implanted in CT‐antibody or specific P38 MAPK inhibitor‐treated uterus was significantly lower than that of the corresponding control, respectively. And the observation of tissue specimen suggested that some embryos were degenerated in CT‐antibody or specific P38 MAPK inhibitor‐treated uterus, and adipose vacuoles were present in the decidual cells. In conclusion, CT promoted the development of pre‐implantation embryos and the intracellular Ca²⁺‐dependent P38MAPK signal molecule was involved in the process.