The yolk sac (YS) consists of the yolk, which supplies nutrients, and the YS tissue, which surrounds the yolk and provides essential metabolic functions for the developing embryo. The YS tissue is derived from the midgut of the embryo and consists of a layer of endodermal epithelial cells (EEC) in contact with the yolk contents, a mesodermal layer that contains the vascular system and an outer ectodermal layer. The YS tissue is a multifunctional organ that provides essential functions such as host immunity, nutrient uptake, carbohydrate and lipid metabolism, and erythropoiesis. The YS tissue plays a role in immunity by the transport of maternal antibodies in the yolk to the embryonic circulation that feeds the developing embryo. In addition, the YS tissue expresses high mRNA levels of the host defense peptide, avian beta-defensin 10 during mid embryogenesis. Owing to its origin, the YS EEC share some functional properties with intestinal epithelial cells such as expression of transporters for amino acids, peptides, monosaccharides, fatty acids, and minerals. The YS tissue stores glycogen and expresses enzymes for glycogen synthesis and breakdown and glucogenesis. This carbohydrate metabolism may play an important role in the hatching process. The mesodermal layer of the YS tissue is the site for erythropoiesis and provides erythrocytes before the maturation of the bone marrow. Other functions of the YS tissue involve synthesis of plasma proteins, lipid transport and cholesterol metabolism, and synthesis of thyroxine. Thus, the YS is an essential organ for the growth, development, and health of the developing embryo. This review will provide an overview of the studies that have investigated the functionalities of the YS tissue at the cellular and molecular levels with a focus on chickens.

Simple Summary In domestic birds, breeding practices and optimisation of the microenviroment and nutrition ensure egg production throughout the entire year. However, domestic geese experience an annual cycle of reproductive quiescence and recrudescence. Thus, patterns of reproductive hormones related to the initiation and termination of the breeding-laying period between the sexes seems to be especially important. This paper presents annual patterns of prolactin (PRL), triiodothyronine (T3), thyroxine (T4), testosterone (T), progesterone (P4), and estradiol (E2) in ganders and female geese. Long-day breeding Zatorska geese kept in controlled commercial conditions experienced periods with elevated plasma PRL levels in both sexes post-breeding and during the second half of the breeding-laying period. Increased plasma PRL levels by the end of the breeding-laying period were detected earlier in ganders than in female geese. Annual patterns of thyroid hormones (THs) were partially in agreement with existing theories on the specific role of THs in termination of breeding, which is permissive rather than causal. It may be suggested that ganders terminate their breeding-laying period one month earlier than female geese. These results may be useful in the manipulation of the endocrine axis to extend the duration of seasonal hatching egg production. This paper examines the dynamics of circulating hormone changes connected with reproduction in geese during the annual period related to gonad morphometry. One hundred geese were examined. The levels of prolactin (PRL), triiodothyronine (T3), thyroxine (T4), testosterone (T), progesterone (P4) and estradiol (E2) were estimated. In both sexes, PRL level patterns fit a quadratic trend with elevations in the post-breeding and the second half of the breeding-laying periods. During these periods, differences in the PRL level between sexes were noted. In ganders, increased PRL levels during the laying period occurred earlier compared to in female geese. Cubic trends for T and E2 in ganders and quadratic for T, P4, and E2 in female geese were observed. PRL was negatively correlated with T in both sexes and with P4 and E2 in female geese. A higher level of T3 and variation in T4 in ganders with a quartic trend in ganders vs. a quadratic in female geese were noted. Patterns of PRL, T, and E2 suggested that the breeding-laying period in ganders may be shorter than in female geese. These findings will be used to explore experimental manipulations of the endocrine axis to increase synchronisation of both sexes.

This study aimed to assess the effects of breeder age on egg quality and amino acid and mineral transfer to the egg yolk and yolk sac of newly hatched chicks. Three ages (32, 42 and 52 weeks) of the same commercial flock of Hubbard breeders were studied. A total of 465 eggs were used for each age, with 60 being used for determining egg quality and amino acid and mineral content of yolk, and 405 for incubation period to obtain and evaluate the yolk sac of chicks. Breeders aged 52 weeks had heavier eggs and a higher percentage of yolk (p < 0.05), whereas 32-week-old breeders had higher eggshell percentage and thickness (p < 0.05). The percentage of protein deposited in egg yolk for 52-week-old breeders was higher than that for 32- and 42-week-old breeders (p < 0.05). Percentages of methionine, cysteine, met + cysteine, lysine, threonine, tryptophan, arginine and isoleucine in egg yolk for 32-week-old breeders were higher than that for 42- and 52-week-old breeders (p < 0.05). The transfer from breeder of phosphorus, potassium, calcium, magnesium, copper, iron, manganese and zinc to the yolk of eggs from 32-week-old breeders was greater than that for eggs from 42- and 52-week-old breeders (p < 0.05). Chicks from 32-week-old breeders had greater deposition of phosphorus and calcium in the yolk sac (p < 0.05). Breeder age did not affect the deposition of potassium, magnesium, copper, iron, manganese and zinc in the yolk sac of newly hatch chicks (p > 0.05). It can, however, be concluded that younger breeders deposit more amino acids and minerals in egg yolk, while embryos of older breeders seem to use the nutrients present in the yolk more efficiently during embryonic development.

Simple Summary:& nbsp;This study presents in silico models of neurokinin B receptor tiTac3Ra (tilapia Tachykinin 3 receptor a) and its potential binding sites, as well as docking native tilapia Neurokinin F (tiNKF) and tilapia Neurokinin B (tiNKB) to theses orthosteric binding sites. For a better understanding of the binding confirmation and interaction of the structures, we compared the conformation between peptide docking and induced fit docking results. We have tried to analyze the affinity of binding and binding site interactions parallel to in-vitro results of receptor activity. NKB antagonists inhibit male tilapia gonadal development and gonadotropin release. We further verified the in-vivo effect of the antagonists on gonadal development in males. Studying the receptor activity of variants with alanine mutations at Phe251(6.44) and Met289(7.43) respectively, we found that, while both variants completely underperformed, there was no direct interaction with the ligand in the binding process indicating their role in post binding receptor activation rather than the binding process itself.
NKB (Neurokinin B) is already known to play a crucial role in fish reproduction, but little is known about the structure and function of NKB receptors. Based on an in silico model of the tilapia NKB receptor Tachykinin 3 receptor a (tiTac3Ra) found in the current study, we determined the key residues involved in binding to tilapia NKB and its functional homologue NKF (Neurokinin F). Despite studies in humans suggesting the crucial role of F251(6.44) and M289(7.43) in NKB binding, no direct peptide interaction was observed in tilapia homologs. In-silico, Ala mutations on residues F251(6.44) and M289(7.43) did not influence binding affinity, but significantly affected the stability of tiTac3Ra. Moreover, in vitro studies indicated them to be critical to tiNKB/tiNKF-induced receptor activity. The binding of NKB antagonists to tiTac3Ra both in-vitro and in vivo inhibits FSH (follicle stimulating hormone) and LH (luteinizing hormone) release and sperm production in mature tilapia males. Non-peptide NKB antagonist SB-222200 had a strong inhibitory effect on the Tac3Ra activation. SB-222200 also decreased LH plasma levels; two hours post intraperitoneal injection, changed sperm volume and the ratios of the different stages along the spermatogenesis in tilapia testes.

Stressful environmental events in early life have long-lasting consequences on later stress responses. We previously showed that heat conditioning of 3-day-old chicks during the critical period of heat-response development leads to heat vulnerability later in life. Here we assessed the role of early-life heat stress on the inflammatory response in the chick anterior hypothalamus (AH), focusing on hypothalamic microglia. We identified the microglial cell population in the chick AH using anti-KUL01 and anti-CD45 antibodies. Specific microglial features were also confirmed by expression of their signature genes. Under normal environmental conditions, hypothalamic microglia isolated from lipopolysaccharide (LPS)-injected chicks displayed a classical activated proinflammatory profile accompanied by a decreased homeostatic signature, demonstrating similarity of immune response with mammalian microglial cells. In accordance with our previous observations, conditioning of 3-day-old chicks under high ambient temperature decreased the number of newborn cells in the AH, among them microglial precursors. Although heat exposure did not affect microglial cell viability, it had a long-term impact on LPS-induced inflammatory response. Exposure to harsh heat led to heat vulnerability, and attenuated recruitment of peripheral monocytes and T cells into the AH following LPS challenge. Moreover, heat conditioning altered microglial reactivity, manifested as suppressed microglial activation in response to LPS. Innate immune memory developed by heat conditioning might underlie suppression of the microglial response to LPS challenge. We describe alterations in genome-wide CpG methylation profile of hypothalamic microglia, demonstrating probable epigenetic involvement in the reprogramming of microglial function, leading to heat-induced inflammatory cross-tolerance.

Targeted in ovo green light (GL) photostimulation during the last days of broiler egg incubation increases embryonic expression of the somatotropic axis, similar to in ovo green light photo stimulation from embryonic day (ED) 0 to the end of incubation. The aim of this study was to examine the effect of selected in ovo GL photostimulation periods on post-hatch broiler growth. Four hundred twenty fertile broiler eggs were divided into 7 treatment groups: the first incubated in the dark (standard conditions) as a negative control; the second incubated under monochromatic GL from ED0-ED20 (positive control); the third group incubated under monochromatic GL light from ED15-ED20; the fourth, fifth and sixth groups were incubated under monochromatic GL on ED16, ED17, and ED18, respectively; and the seventh group was incubated under monochromatic GL from ED18-ED20. All illumination was provided intermittently using LED lamps. After hatch, all chicks were transferred to a controlled room under standard rearing conditions. The group incubated under green light from ED18 until hatch showed similar results to the positive control group in body weights, as well as breast muscle weights (as % of body weights), and an elevation in the somatotropic axis activity during the experiment. We suggest that broiler embryos can be exposed to in ovo GL photostimulation from ED18 until hatch (hatching period), and still exhibit the same performance as obtained by photostimulation from d 0 of incubation.

Targeted green light photostimulation during the last stage of broiler incubation increases expression of the somatotropic axis. The purpose of this study was to further shorten the in ovo green light photostimulation and determine the critical age for photo stimulation in broilers embryos, as a future strategy for broiler incubation. Fertile broilers eggs (n = 420) were divided into 5 treatment groups. The first group was incubated under standard conditions (in the dark) as the negative control group. The second was incubated under intermittent monochromatic green light using light emitting diode lamps with an intensity of 0.1 W/m(2) at shell level from embryonic day (ED) 0 of incubation until hatch, as a positive control. The third, fourth, and fifth groups were incubated under intermittent monochromatic green light from ED 15, 16, and 18 of incubation, respectively, until hatch. All treatment groups showed elevated somatotropic axis expression compared with the negative control, with the group incubated under monochromatic green light from ED 18 until hatch showing results closest to the positive control. This suggests that broiler embryos can be exposed to in ovo green light photostimulation from a late stage of incubation (when transferring the eggs to the hatchery) and exhibit essentially the same outcome as obtained by photostimulation during the entire incubation period.

We evaluated the effects of processing - pasteurization and yoghurt manufacturing - on some health-promoting lipidome components in milk from two feeding treatments - brushland grazing or hay-feeding in confinement - in dairy goats. The contents of fat and protein were higher, and of urea, lower, in grazing goats. Fatty acid composition - at the exception of saturated fatty acids - was affected by dietary management and milk processing. Phospholipid contents was lower in confined goats, with little effect for processing. The phospholipid-totriglyceride ratio was decreased by pasteurization. Sensitivity to pasteurization of phospholipid composition differed between feeding treatments. The percentage of sphingomyelin increased following pasteurization, with no response for fermentation to yoghurt. These results can be exploited to modulate health-promoting fats in dairy products.

Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIFI A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metform in, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs. [GRAPHICS] .

BackgroundMaintenance of the corpus luteum (CL) beyond the time of luteolysis is essential for establishing pregnancy. Identifying the distinct features of early pregnancy CL remains unresolved, hence we analyzed here the transcriptome of CL on day 18 pregnant (P) and non-pregnant (NP) cows using RNA-Seq. CL of P cows expressed ISGs, verifying exposure to the pregnancy recognition signal, interferon-tau (IFNT), whereas the CL of NP cows had elevated luteal progesterone levels, implying that luteolysis had not yet commenced.ResultsThe DEGs, IPA, and metascape canonical pathways, along with GSEA analysis, differed markedly in the CL of P cows from those of NP cows, at the same day of the cycle. Both metascape and IPA identified similar significantly enriched pathways such as interferon alpha/beta, sonic hedgehog pathway, TNFA, EDN1, TGFB1, and PDGF. However, type-1 interferon and sonic hedgehog pathways were positively enriched whereas most of the enriched pathways were downregulated in the P compared to NP samples. Thirty-four % of these pathways are known to be elevated by PGF2A during luteolysis. Notably, selective DEGs in luteinized granulosa cells were modulated by IFNT in vitro in a similar manner to their regulation in the CL of P cows.ConclusionThis study unraveled the unique transcriptomic signature of the IFNT-exposed, early pregnancy CL, highlighting the abundance of downregulated pathways known to be otherwise induced during luteolysis. These and IFNT-regulated in vitro pregnancy-specific DEGs suggest that IFNT contributes to the characteristics and maintenance of early pregnancy CL.

The ovarian pool of follicles, and their enclosed oocytes, is highly sensitive to hyperthermia. Heat-induced changes in small antral follicles can later manifest as impaired follicle development and compromised competence of the enclosed oocytes to undergo maturation, fertilisation and further development into an embryo. This review describes the main changes documented so far that underlie the oocyte damage. The review discusses some cellular and molecular mechanisms by which heat stress compromises oocyte developmental competence, such as impairment of nuclear and cytoplasmic maturation and mitochondrial function, changes in the expression of both nuclear and mitochondrial transcripts and the induction of apoptosis. The review emphasises that although the oocyte is exposed to heat stress, changes are also evident in the developed embryo. Moreover, the effect of heat stress is not limited to the summer; it carries over to the cold autumn, as manifest by impaired steroid production, low oocyte competence and reduced fertility. The spontaneous recovery of oocytes from the end of the summer through the autumn until the beginning of winter suggests that only subpopulations of follicles, rather than the entire ovarian reserve, are damaged upon heat exposure.

Artificial targeted illumination has a pivotal role in reproductive processes of poultry. The light-absorption mechanism in birds consists of 2 main components: the eye (retinal photoreceptors) and extraretinal photoreceptors located in the brain. Previous studies conducted on hens have shown that photostimulation of brain extraretinal photoreceptors elevates reproductive activity, whereas retinal photostimulation suppresses it. We tested the effect of targeted differential photostimulation (TDP) on reproductive activities of broiler breeder males. Fifty broiler breeder roosters (Ross), 21 wk of age, were divided into 5 environmentally controlled light-treatment rooms (n = 10) equipped with individual cages. Rooms 1 and 2 had 2 parallel lighting systems consisting of red light (630 nm) and green light (514 nm), and rooms 3 and 4 had parallel red and blue (456 nm) lighting systems. Room 5, illuminated with white light, served as the control. Birds of all groups were kept under short day (6L:18D) for 2 wk with both lighting systems in each treatment room turned on. At 23 wk of age, birds were photostimulated by gradually increasing one of the lighting systems to 14 h of light in each room, while the other lighting system was left on short day (6L:18D). Weekly semen samples were collected until 65 wk of age and analyzed for volume, motility, concentration and vitality. Monthly blood samples were drawn for plasma hormone assays. At 65 wk of age, roosters were euthanized and hypothalamus, pituitary gland, retina and testes samples were taken for mRNA expression analysis. TDP using long-day red light and short-day green light significantly increased reproductive performance, manifested by higher semen volume, motility and concentration, and testis weight; furthermore, this group had higher plasma testosterone levels, higher GnRH mRNA expression in the hypothalamus, lower levels of aromatase in the testes, and lower mRNA expression of hypothalamic serotonin transporter, and of pituitary prolactin and its receptors in the testes. This is the first study showing a positive effect of TDP on reproduction of broiler breeder roosters.

Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1 mu M) or DACT (1 or 10 mu M) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42 h and 7 days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV-) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV-) and DACT (AV+, AV-) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV- vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo. Summary sentence Summary sentence: Atrazine and its metabolite, diaminochlorotriazine, induce multifactorial damage in sperm, which is carried over to the embryo's transcriptome.

Hydrophobic carriers were examined for geosmin and 2-methylisoborneol removal from water derived from an aquaculture system. A combination of adsorption and biodegradation was found to underlie the removal of the off-flavor compounds. Adsorption of these compounds by the carriers was unaffected by the presence of organic matter in the water to be treated. A model based on adsorption/desorption and first-order degradation kinetics provided an accurate prediction for experimentally determined 2-methylisoborneol removal rates. Steady removal of geosmin and 2-methylisoborneol as well as nitrate reduction were observed during long-term operation of the plug-flow reactors with water derived from an aquaculture facility. Metagenomic analysis of the microbial community on the carriers during long-term operation of the reactors revealed a predominance of denitrifying bacteria. It was found that geosmin and 2-methylisoborneol led to statistically significant changes in the abundances of 21 contigs that contained genes involved in terpene degradation. This study shows that at low ambient concentrations of geosmin and 2-methylisoborneol in nitrate and organic-rich water, such as found in aquaculture systems, their biodegradation can be accomplished by terpene-degrading denitrifiers that develop on hydrophobic carriers used for filtration of the contaminated water.

Simple Summary We used an automatic activity-monitoring system to determine onset of estrus in dairy cows. Within 5 h of onset, we administered a single injection of GnRH analogue to improve fertility during the summer and autumn. The treatment increased pregnancy per insemination during the autumn, but not in the summer. The subgroups for which the treatment specifically tended to improve conception risk during the autumn were: mature (2nd plus parity) cows and cows with uterine disease and ketosis after calving. Detection of estrus onset by activity monitoring and GnRH administration shortly thereafter could be incorporated into a synchronization program, to improve fertility of positively-responding subpopulations of cows. We examined gonadotropin-releasing hormone (GnRH) administration at onset of estrus (OE), determined by automatic activity monitoring (AAM), to improve fertility of dairy cows during the summer and autumn. The study was performed on two dairy farms in Israel. The OE was determined by AAM recorded every 2 h, and a single im dose of GnRH analogue was administered shortly after OE. Pregnancy was determined by transrectal palpation, 40 to 45 d after artificial insemination (AI). Conception risk was analyzed by the GLIMMIX procedure of SAS. Brief visual observation of behavioral estrus indicated that about three-quarters of the events (n = 40) of visually detected OE occurred within 6 h of AAM-detected OE. Accordingly, the GnRH analogue was administered within 5 h of AAM-detected OE, to overlap with the expected endogenous preovulatory LH surge. Overall, pregnancy per AI (P/AI) was monitored over the entire experimental period (summer and autumn) in 233 first, second or third AI (116 and 117 AI for treated and control groups, respectively). Least square means of P/AI for treated (45.8%) and control (39.4%) groups did not differ, but group-by-season interaction tended to differ (p = 0.07), indicating no effect of treatment in the summer and a marked effect of GnRH treatment (n = 58 AI) compared to controls (n = 59 AI) on P/AI in the autumn (56.6% vs. 28.5%, p < 0.03). During the autumn, GnRH-treated mature cows (second or more lactations), and postpartum cows exhibiting metabolic and uterine diseases, tended to have much larger P/AI than their control counterparts (p = 0.07-0.08). No effect of treatment was recorded in the autumn in first parity cows or in uninfected, healthy cows. In conclusion, administration of GnRH within 5 h of AAM-determined OE improved conception risk in cows during the autumn, particularly in those exhibiting uterine or metabolic diseases postpartum and in mature cows. Incorporation of the proposed GnRH treatment shortly after AAM-detected OE into a synchronization program is suggested, to improve fertility of positively responding subpopulations of cows.

Simple Summary:& nbsp;Spermatozoa move forward in the female reproductive tract in a linear pattern defined as progressive motility (PM). PM is associated with fertilization competence and used as a parameter to evaluate the spermatozoa's quality. The livestock-breeding industry is based on artificial insemination with cryopreserved spermatozoa, making the relationship between PM and cryosurvival important. This study explores the association between PM and spermatozoa's quality, cryotolerance and fertilization competence. The number of progressively motile spermatozoa within a standard insemination straw positively correlates with conception rate, making progressively motile spermatozoa's survival during cryopreservation a potentially relevant parameter for spermatozoa evaluation.
An association between progressive motility (PM) and spermatozoa fertility competence has been suggested. However, the mechanism that underlies PM is not clear enough. We examined physiological characteristics and fatty acid composition of fresh spermatozoa with high and low PM. Additional analysis of fatty acid composition and structural characteristics was performed on spermatozoa samples with high and low progressively motile spermatozoa's survival (PMSS), i.e., the ratio between the proportion of progressively motile spermatozoa after and before cryopreservation. Finally, a fertility field trial was conducted to examine the association between the number of PM spermatozoa within the insemination straw post thawing and conception rate. Analysis of fresh spermatozoa revealed a higher omega-6 to omega-3 ratio in ejaculates with low PM relative to those with high PM (p < 0.01). The proportion of polyunsaturated fatty acids was higher in low-PMSS fresh samples (p < 0.05) relative to their high-PMSS counterparts. Fresh samples with high-PMSS expressed a higher mitochondrial membrane potential (p < 0.05) and a higher proportion of viable cells that expressed reactive oxygen species (ROS; p < 0.05). Post-thawing evaluation revealed a reduced proportion of progressively motile sperm, with a prominent effect in samples with high PM relative to low PM, defined before freezing (p < 0.01). No differences in spermatozoa mitochondrial membrane potential or ROS level were found post-thawing. A fertility study revealed a positive correlation between the number of progressively motile spermatozoa within a standard insemination straw and conception rate (p < 0.05). Considering these, the bull PMSS is suggested to be taken into account at the time of straw preparation.

Microvilli generate the small intestinal brush border, the main site of nutrient digestion and absorption. Mucosal structuring of the small intestine of chicken during the perihatch period has been widely researched, yet the developmental dynamics of micro-villi during this period have not been fully character-ized. In this study, we examined the structural and molecular characteristics of microvilli assembly and maturation during the perihatch period. Small intes-tines of broiler embryos and chicks were sampled at prehatch ages 17 E and 19 E, at day of hatch (DOH) and at 1, 3, 7, and 10 d posthatch. Morphological evalu-ations and measurements were conducted by scanning electron microscopy (SEM) and light microscopy (LM) (n = 3/timepoint), and expression of microvilli structural genes Plastin 1, Ezrin, and Myo1a was exam-ined by Real-Time qPCR (n = 6/timepoint). Results revealed dissimilar patterns of microvilli and villi devel-opment during the perihatch period. From 19 E to 1 d, microvilli lengths increased 4.3-fold while villi lengths increased 2.8-fold (P < 0.0001). From 3 to 7 d, villi lengths increased by 20% (P < 0.005), while microvilli lengths decreased by 41% (P = 0.001). At 10 d, micro-villi lengths stabilized, while villi continued to elongate by 26% (P < 0.0001). Estimations of the microvilli amplification factor (MAF) and total enterocyte surface area (TESA) revealed similar trends, with peak values of 78.53 and 1961.67 mu m(2), respectively, at 3 d. Microvilli structural gene expression portrayed diverse patterns. Expression of Plastin 1, which bundles and binds actin cores to the terminal web, increased 8.7-fold between 17 E and DOH (P = 0.005), and gradually increased up to 7 d (P = 0.045). Ezrin and Myo1a, both actin core-cell membrane cross-linkers, portrayed dif-ferent expression patterns throughout the perihatch period, as Ezrin expression was relatively stable, while Myo1a expression increased 15.8-fold between 17 E and 10 d (P < 0.0001). We conclude that microvilli assem -bly during the perihatch period is a rapid, coordinated process, which dramatically expands the digestive and absorptive surface area of the small intestine before the completion of villi maturation.

Spexin (SPX), a neuropeptide evolutionarily conserved from fish to mammals, is widely distributed in the brain and peripheral tissues and associated with various physiological functions. Recently SPX has been suggested to be involved in neurological mechanism of stress. The current study investigates the involvement of SPX in chronic social defeat stress, using male teleost, the Nile tilapia (Oreochromis niloticus) as an animal model due to its distinct social hierarchy of dominant and subordinate relationship. The tilapia genome has SPX1a and SPX1b but has no SPX2. In the Nile tilapia, we localized SPX1a and SPX1b in the brain using in-situ hybridization. Next, using qPCR we examined gene expression of SPX1a and SPX1b in chronically stress (socially defeated) fish. SPX1a expressing cells were localized in the semicircular torus of the midbrain region and SPX1b expressing cells in the telencephalon. Chronically stress fish showed elevated plasma cortisol levels; with an upregulation of SPX1a and SPX1b gene expression in the brain compared to non-stress (control) fish. Since social defeat is a source of stress, the upregulated SPX mRNA levels during social defeat suggests SPX as a potentially inhibitory neuropeptide capable of causing detrimental changes in behaviour and physiology.

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary-gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques-clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)-were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.