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Carotenoids are among the best-known pigments in nature, confer color to plants and animals, and are mainly derived from photosynthetic bacteria, fungi, algae, plants. Mammals cannot synthesize carotenoids. Carotenoids' source is only alimentary and after their assumption, they are mainly converted in retinal, retinol and retinoic acid, collectively known also as pro-vitamins and vitamin A, which play an essential role in tissue growth and regulate different aspects of the reproductive functions. However, their mechanisms of action and potential therapeutic effects are still unclear. This review aims to clarify the role of carotenoids in the male and female reproductive functions in species of veterinary interest. In female, carotenoids and their derivatives regulate not only folliculogenesis and oogenesis but also steroidogenesis. Moreover, they improve fertility by decreasing the risk of embryonic mortality. In male, retinol and retinoic acids activate molecular pathways related to spermatogenesis. Deficiencies of these vitamins have been correlated with degeneration of testis parenchyma with consequent absence of the mature sperm. Carotenoids have also been considered anti-antioxidants as they ameliorate the effect of free radicals. The mechanisms of action seem to be exerted by activating Kit and Stra8 pathways in both female and male. In conclusion, carotenoids have potentially beneficial effects for ameliorating ovarian and testes function.

The objectives of this study were to evaluate in horse testes the expression of kisspeptin (KiSS) and GnRH1 neuropeptides and their cognate receptors, KiSS1R and GnRH1R, as well as their action on testosterone, GnRH1, prostaglandin F2α (PGF2α), and PGE2 synthesis and cyclooxygenase 1 (COX1) and COX2 activity by Leydig cells in vitro. Testes were obtained from 9 sexually mature horses by surgical castration. Immunohistochemistry, evidenced the presence of KiSS, KiSS1R, GnRH, and GnRH1R in Leydig cells, whereas germinal and Sertoli cells were positive only for GnRH1. Transcripts for both neuropeptides and their cognate receptors were revealed in isolated Leydig cells by RT-PCR. Isolated and purified Leydig cells were in vitro cultured with agonists and antagonists of KiSS (KiSS-10 and KiSS-234, respectively) and GnRH1 (buserelin and antide, respectively). KiSS-10 and buserelin increased (P 0.01) COX1 activity and testosterone and PGF2α basal secretion, while decreased (P 0.01) that of PGE2. KiSS-10 and buserelin did not affect COX2 activity. GnRH1 basal production was increased (P 0.01) by KiSS-10, but not by buserelin. Antide counteracted the KiSS and GnRH1 effects, whereas KiSS-234 influence only those of KiSS. Summarizing, the KiSS/GnRH1 system is present in horse Leydig cells and modulates their endocrine activity. In particular, the endocrine effects of KiSS are mediated by GnRH1, so suggesting that hypothalamic-like interaction between KiSS and GnRH1 occurs also in Leydig cells.

Stimuli that lead to the release of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropins and, consequently, ovulation in mammals fall into two broad categories. In the first, high plasma oestrogen concentrations induce the events that trigger ovulation, a characteristic of spontaneous ovulators. In the second, nerve stimuli occurring during mating reach the hypothalamus and trigger the release of GnRH and ovulation with a neuroendocrine reflex that characterizes induced ovulators.In this review, we will give an overview of the distribution of NGF and its expression in the different tissues of the male accessory sex glands, the main sites of NGF production. Next, we will highlight the role of NGF in sperm function and its potential cryopreserving role in artificial insemination techniques. Finally, we will evaluate the functions of NGF in ovulation, particularly in induced ovulators. Overall, the information obtained so far indicates that NGF is widely distributed in organs that regulate the reproductive activity, in both males and females. In spontaneous ovulators, NGF exerts mainly a luteotrophic action, while, in induced ovulators it is the main ovulation-inducing factor. A better understanding of the role of NGF in reproduction would be of great interest, since it could help finding innovative therapeutic aids to improve mammalian fertility.

Stimuli that lead to the release of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropins and, consequently, ovulation in mammals fall into two broad categories. In the first, high plasma oestrogen concentrations induce the events that trigger ovulation, a characteristic of spontaneous ovulators. In the second, nerve stimuli occurring during mating reach the hypothalamus and trigger the release of GnRH and ovulation with a neuroendocrine reflex that characterizes induced ovulators.In this review, we will give an overview of the distribution of NGF and its expression in the different tissues of the male accessory sex glands, the main sites of NGF production. Next, we will highlight the role of NGF in sperm function and its potential cryopreserving role in artificial insemination techniques. Finally, we will evaluate the functions of NGF in ovulation, particularly in induced ovulators. Overall, the information obtained so far indicates that NGF is widely distributed in organs that regulate the reproductive activity, in both males and females. In spontaneous ovulators, NGF exerts mainly a luteotrophic action, while, in induced ovulators it is the main ovulation-inducing factor. A better understanding of the role of NGF in reproduction would be of great interest, since it could help finding innovative therapeutic aids to improve mammalian fertility.

Simple Summary Corpora lutea (CL) are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. A variety of regulatory factors come into play in modulating the functional lifespan of CL, with luteotropic and luteolytic effects. Many aspects of luteal phase physiology have been clarified, yet many others have not yet been determined, including the molecular and/or cellular mechanisms that maintain the CL from the beginning of luteolysis during early CL development. This paper summarizes our current knowledge of the endocrine and cellular mechanisms involved in multifactorial CL lifespan regulation, using the pseudopregnant rabbit model. Abstract Our research group studied the biological regulatory mechanisms of the corpora lutea (CL), paying particular attention to the pseudopregnant rabbit model, which has the advantage that the relative luteal age following ovulation is induced by the gonadotrophin-releasing hormone (GnRH). CL are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. It is now clear that, besides the classical regulatory mechanism exerted by prostaglandin E2 (luteotropic) and prostaglandin F2α (luteolytic), a considerable number of other effectors assist in the regulation of CL. The aim of this paper is to summarize our current knowledge of the multifactorial mechanisms regulating CL lifespan in rabbits. Given the essential role of CL in reproductive success, a deeper understanding of the regulatory mechanisms will provide us with valuable insights on various reproductive issues that hinder fertility in this and other mammalian species, allowing to overcome the challenges for new and more efficient breeding strategies.

The aim of this study was to evaluate the effect of dietary polyphenols on the expression of the effectors involved in inflammation and apoptosis in rabbit ovary. New Zealand White female rabbits were fed a basal control diet (CTR), or the same diet supplemented with a polyphenolic concentrate (POL, 282.4 mg/kg) obtained from olive mill waste waters. The follicle counts and the relative mRNA (RT-qPCR) and protein (immunohistochemistry) expression of the effectors involved in inflammation (cyclooxygenase-2, interleukin-1beta, tumor necrosis factor-alpha, TNFA) and apoptosis (BCL2-associated X protein, BAX), detected in the ovaries of both groups, were examined. The POL diet increased the primary and total follicles number. Cyclooxygenase-2 gene expression was higher (p &lt, 0.05) in the POL group than in the CTR group, whereas BAX was lower (p &lt, 0.05) in POL than CTR. Immunohistochemistry revealed the presence of all the proteins examined, with weaker (p &lt, 0.05) COX2 and BAX signals in POL. No differences between the CTR and POL groups were observed for IL1B and TNFA gene and protein expression. These preliminary findings show that dietary polyphenols modulate inflammatory and apoptotic activities in rabbit ovary, regulating cyclooxygenase-2 and BAX expression, thus suggesting a functional involvement of these dietary compounds in mammalian reproduction.

Simple Summary Invasive alien species pose a significant threat to biodiversity, as once they have adapted to their new environment, they cause the reduction and even extinction of native species. In this framework, the American grey squirrel (Sciurus carolinensis) poses a serious threat to the European red species squirrel (Sciurus vulgaris), especially in the Umbria region of Italy. In fact, an invasive grey squirrel population has adapted well to the Umbrian territory, showing high reproductive success. In addition to its role in the development of the vertebrate nervous system, nerve growth factor (NGF) has recently been found to play an important role in reproduction. In order to investigate the reproductive physiology of female grey squirrels, the ovarian presence, distribution, and gene expression of NGF and its cognate receptors were evaluated during both breeding and nonbreeding seasons. The presence and gene expression of this system at the ovarian level, mainly during the breeding season, confirm the possible involvement of NGF and its receptors in the gonadal activity of this invasive grey squirrel population. Abstract The grey squirrel is an invasive alien species that seriously threatens the conservation of the native red squirrel species. With the aim of characterizing the reproductive physiology of this species due to its great reproductive success, the function of the ovarian nerve growth factor (NGF) system was analyzed in a grey squirrel population living in central Italy. During the breeding and nonbreeding seasons, the ovarian presence, distribution, and gene expression of NGF, neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR), as well as NGF plasma concentrations, were evaluated in female grey squirrels. NGF was found in the luteal cells and in the thecal and granulosa cells of follicles, while NTRK1 and NGFR were only observed in follicular thecal and granulosa cells. NGF and NGFR transcripts were almost two-fold greater during the breeding season, while no seasonal differences were observed in NTRK1 gene expression. During the breeding season, NGFR was more expressed than NTRK1. Moreover, no changes were observed in NGF plasma levels during the reproductive cycle. The NGF system seems to be involved in regulating the ovarian cycle mainly via local modulation of NGF/NGFR, thus playing a role in the reproductive physiology of this grey squirrel population.

Resveratrol is one of the most investigated natural polyphenolic compounds and is contained in more than 70 types of plants and in red wine. The widespread interest in this polyphenol derives from its antioxidant, anti-inflammatory and anti-aging properties. Several studies have established that resveratrol regulates animal reproduction. However, the mechanisms of action and the potential therapeutic effects are still unclear. This review aims to clarify the role of resveratrol in the male and female reproductive functions, with a focus on animals of veterinary interest. In the female, resveratrol has been considered a phytoestrogen due to its capacity to modulate ovarian function and steroidogenesis via sirtuins, SIRT1, in particular. Resveratrol has also been used to enhance aged oocyte quality, and as a gametes cryo-protectant with mainly antioxidant, and anti-apoptotic effects. In the male, resveratrol enhanced testes function and spermatogenesis through activation of AMPK pathway. Furthermore, resveratrol has been supplemented to semen extenders improving the preservation of sperm quality. In conclusion, resveratrol has potentially beneficial effects for ameliorating ovarian and testes function. However, due to unclear data, further studies are necessary to consolidate these findings.

Aglepristone was administered in bitches during the follicular phase to evaluate its effects on progesterone, estradiol-17β and LH serum concentrations. Ten German Shepherds were divided into two groups (treated n=5; control n=5). Treated bitches received 10mg/kg BW of aglepristone subcutaneously during the early follicular phase, 24hr after and then 7days later. The control group was injected, at the same time periods, with saline solution (0.3ml/kg BW). For the steroid evaluations, blood was collected daily from the onset of proestrus until the first day of cytological dioestrus. For LH base-line serum determination, blood was also collected every 20min for 2hr at the onset of proestrus. For LH surge identification, blood was collected daily (every 6hr) starting from the day of the first administration of aglepristone or saline solution until the first day of dioestrus. All animals ovulated but the treated group presented longer ovulation-dioestrus intervals than the control group (5.2±2.2days p&nbsp

Apelin is a potent inotropic agent causing endothelium-mediated vasodilation and is involved in vessel formation by interacting with a specific receptor. Its cardiovascular profile suggests a role in the regulation of gestational hemodynamic changes. The expression of apelin and its receptor has been reported in some portions of the reproductive tract of different mammalian species. As far as we know, there are no reports describing the expression of apelin and apelin receptor in bitch’s placenta. Therefore, the aim of this study was to investigate, for the first time, the presence and distribution of apelin and apelin receptor in bitch placenta by molecular biology and immunohistochemical techniques. Sixteen adult female half-breed bitches were used. The animals were divided into two groups based on the stage of pregnancy: group 1 (mid-gestation n = 8) and group 2 (end gestation n = 8). These bitches were subjected to ovariohysterectomy (group1) or non-conservative caesarean section (group 2). The immunohistochemical technique revealed the presence of positive immune reaction for apelin and apelin receptor in all the samples examined at 30 days and at the end of pregnancy. In particular, apelin and apelin receptor staining was evident in the cytoplasms of cytotrophoblasts and in epithelial cells of the maternal portion. Even if not included into the structure of the placenta, the uterine glands also exhibited a positive immune reaction for apelin and apelin receptor. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all the placenta samples examined. On the basis of our results it was also possible to hypothesize a potential role of apelin in the control of local placenta blood flow during pregnancy development in bitches.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

To investigate the ovulatory mechanisms triggered by raw semen (RS) in rabbits, we examined the expression of nerve growth factor (NGF)—a supposed ovulation-inducing factor (OIF)—and cognate receptors in anterior pituitary, ovary, and cervix as well as plasma NGF and luteinizing hormone (LH) concentrations. Six does/group were sham-inseminated with sterile saline (PBS), naturally mated (NM), inseminated with RS alone or after lumbar anesthesia (ARS), or treatment with COX inhibitors (CIRS). Immunohistochemistry revealed positive signals for NGF and receptors in all tissues. RT-PCR confirmed the presence of the target transcripts in the same tissues, except NTRK1 in the cervix. Circulating NGF concentrations rose 3- to 6-fold (P < 0.01) 15 min after semen deposition into the genital tract of NM, RS, and ARS rabbits and remained sustained thereafter. Circulating NGF was 4-fold lower (P < 0.01) in CIRS than in RS does indicating that NGF is mainly synthesized by the uterus. A concomitant rise of LH and NGF concentrations was found in 83.3%, 50.0%, and 16.7% of NM, RS, and CIRS does, respectively, but not in ARS (despite high NGF circulating levels). Seminal plasma NGF concentration was 151.9 ± 9.25 μg/mL. The ovulatory responses were 0%, 83.3%, 66.7%, 16.7%, and 0% in PBS, NM, RS, ARS, and CIRS groups, respectively. Present data confirm that, although RS may induce ovulation via endocrine mechanisms through binding to NGF receptors in the ovary, a novel OIF-mediated neural mechanism facilitates ovulation in rabbits.

Simple Summary The wastes obtained from olive oil processing have a negative impact on the environment, but are rich in bioactive molecules such as phenolic compounds. These compounds have recently been used to manufacture nutritional supplements to improve animal health and welfare, productive performances, and to produce functional foods of animal origin (meat, milk, cheese). Polyphenols have antioxidant, antimicrobial and anti-inflammatory properties that modulate apoptotic pathways, cytokines, genes and protein expressions in various cellular systems. The liver is the main metabolic organ and several papers have demonstrated that it is a target organ of polyphenol molecules. The aim of this study was to highlight the effects on gene expression of inflammatory, metabolic and apoptotic effectors in the livers of rabbits fed with a polyphenolic concentrate obtained from olive mill waste waters (a residue of the extraction process of extra virgin olive oil). Quantitative Real-Time PCR results showed the down-regulation of SIRT1, TNFA, AGER, BAX and PPARA gene expressions in the POL group compared to the CTR group. These results show for the first time that using olive mill waste waters can prevent the harmful effects of oxidative stress in the cellular systems of food-producing animals such as rabbits. Abstract Agro-industrial processing for the production of food or non-food products generates a wide range of by-products and residues rich in bioactive compounds including polyphenols. The concentration of these by-products is sometimes higher than in the original raw material as in the case of olive mill waste water (OMWW), one of the main by-products of olive oil extraction. Polyphenols are secondary plant metabolites that regulate the expression of specific inflammatory genes, transcriptional factors and pro/anti-apoptotic molecules, thus modulating the signaling pathways essential for cell health and homeostasis. The liver plays a key role in regulating homeostasis by responding to dietary changes in order to maintain nutritional and physiological states. In this study a nutrigenomic approach was adopted, which focuses on the effects of diet–health–gene interactions and the modulation of cellular processes, in order to evaluate the expression of the genes (AGER, BAX, COX2, IL1B, PPARA, PPARG, SIRT1, TNFA) involved in these interactions in the livers of rabbits fed with a diet supplemented with OMWW (POL) or without supplements (control, CTR). The RT-qPCR analysis showed the down-regulation of SIRT1, TNFA, AGER, BAX and PPARA transcripts in the POL group compared to the CTR group. These results show that OMWW dietary supplementation prevents cell death and tissue deterioration in rabbits.

The present research used immunohistochemistry to analyse the detection and localisation of nitric oxide synthase (NOS) isoforms in the ductuli efferentes and epididymis of prepubertal and adult alpaca. In the ductuli efferentes and epididymis of prepubertal and adult animals, nNOS and eNOS were similarly expressed in epithelial lining cells, conversely differences were observed in the immunopresence of iNOS. Our data provide evidence that NOS isoforms may have roles in reproductive functions and in the developmental processes of the excurrent duct system in the alpaca.

Kisspeptin (KiSS) and its related receptors (KiSS1R) have a critical role in the reproduction of mammals. The KiSS/KiSS1R system is expressed in numerous reproductive organs including the ovary. Here, we studied the expression of the KiSS/KiSS1R system and its functional role in rabbit corpora lutea (CL) at days 4 (early-), 9 (mid-), and 13 (late-stage) of pseudopregnancy. In vitro progesterone, prostaglandin (PG) F2α (PGF2α) and E2 (PGE2) productions and prostaglandin-endoperoxide synthase 1 (PTGS1) and 2 (PTGS2) activities were evaluated. Immune reactivity (IR) for KiSS and KiSS1R were detected in luteal cells at nuclear and cytoplasmic level at all luteal stage for KiSS and only at early- and mid-stage for KiSS1R; IR decreased from early- to later stages of pseudopregnancy. The KiSS-10 augmented progesterone and PGE2 and diminished PGF2α secretions by early- and mid-CL; KiSS-10 reduced PTGS2 activity at early- and mid-stages, but did not affect PTGS1 at any luteal stages. The antagonist KiSS-234 counteracted all KiSS-10 effects. This study shows that the KiSS/KiSS1R system is expressed in CL of pseudopregnant rabbits and exerts a luteotropic action by down-regulating PTGS2, which decreases PGF2α and increases PGE2 and progesterone.

The aim of the present study was to evaluate: (1) the presence of nerve growth factor (NGF), neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR) in the rabbit uterus; and (2) the in vitro effects of NGF on PGF2α and PGE2 synthesis and on the PGE2-9-ketoreductase (PGE2-9-K) activity by the rabbit uterus. Nerve growth factor, NTRK1, and NGFR were immunolocalized in the luminal and glandular epithelium and stroma cells of the endometrium. reverse transcriptase polymerase chain reaction indicated the presence of messenger RNA for NGF, NTRK1, and NGFR in the uterus. Nerve growth factor increased (P < 0.01) in vitro secretions of PGF2α and PGE2 but coincubation with either NTRK1 or oxide nitric synthase (NOS) inhibitors reduced (P < 0.01) PGF2α production and blocked (P < 0.01) PGE2 secretion. Prostaglandins releases were lower (P < 0.01) than control when uterine samples were treated with NGF plus cyclooxygenase inhibitor. However, addition of NGFR inhibitor reduced (P < 0.01) PGF2α secretion less efficiently than NTRK1 or NOS inhibitors but had no effect on PGE2 yield. Nerve growth factor increased (P < 0.01) the activity of PGE2-9-K, whereas coincubation with NTRK1 or NOS inhibitors abolished (P < 0.01) this increase in PGE2-9-K activity. However, cotreatment with either cyclooxygenase or NGFR inhibitors had no effect on PGE2-9-K activity. This is the first study to document the distribution of NGF/NTRK1 and NGFR systems and their effects on prostaglandin synthesis in the rabbit uterus. NGF/NTRK1 increases PGF2α and PGE2 productions by upregulating NOS and PGE2-9-K activities, whereas NGF/NGFR augments only PGF2α secretion, through an intracellular mechanism that is still unknown.

The reproductive cycle of an invasive alien Italian grey squirrel population was studied to understand its adaptation and limit its spread, in order to conserve the autochthonous red squirrel. Female and male genital traits were evaluated throughout the reproductive cycle, including the ovary, uterus, testicle, epididymis, seminiferous tubule morphometry, and germinative epithelium histology. Moreover, individual female fecundity was determined by counting uterine scars. Ovary width and uterus weight, length, and width reached their highest values in the luteal and pregnancy phases. On conducting a histological evaluation of the testicular germinal epithelium, four morphotypes related to the different reproductive phases of the male squirrels were identified: immature, pubertal, spermatogenesis, and regressive. Testicle and epididymis weights and seminiferous tubule diameters reached their largest values during spermatogenesis. Uterine scar analysis showed that 69% of the females had given birth to one or two litters, while 31% had no uterine scars. Litters were larger in the first breeding period than in the second, annual fecundity was 4.52 &plusmn, 1.88 uterine scars/female. Umbrian grey squirrels have adapted to their non-native range, showing two annual mating periods at times similar to those in their native range, and high reproductive success.

Kisspeptin and its receptor KISS1R are involved in the neuroendocrine regulation of mammalian reproduction and their role on follicular development and function can be hypothesized. The present work was designed to confirm the immunopresence of kisspeptin and its receptor in the ovary of swine and to study the effects of kisspeptin 10 and its antagonist, kisspeptin 234, on main functional parameters of granulosa cells (i.e. cell proliferation, steroid production, and redox status) as well as their modulatory action on angiogenesis. The immunopresence of kisspeptin and KISS1R were detected in granulosa cells. Kisspeptin 10 stimulated progesterone in vitro production, thus indirectly suggesting that it can have a role in the luteinization process of granulosa cells. Kisspeptin 10 displayed potentiating effects on non-enzymatic scavenging activity, thus supporting its involvement in the control of the antioxidant defense system of ovarian follicles. In addition, results from the angiogenesis bioassay suggest that kisspeptin may have a role in the physiological development of new ovarian vessels. Additional studies are needed to confirm the functional significance of the kisspeptin/KISS1R system within the swine ovary.

Adequate fat diet supplementation shows variable positive effects in farm animal breeding. Omega-3 and n-6 PUFAs are able to modulate several reproductive effectors: the luteolytic PGF2α, the luteotropic PGE2, the nuclear receptor PPARG, and steroids such as E2 and P4. PUFA supplementation favours fertility, onset of estrus, embryo survival, and also parturition by reducing preterm labour risk. These effects are likely mediated by the balance modulation of PGF2α and PGE2 productions, the syntheses of E2 and P4, and the activation of PPARG. As regards to male fertility, the effects of n-3 or n-6 PUFA supplementation at high concentrations in the diet are relatively unknown. PUFAs confer to the spermatozoa plasma membrane the fluidity it needs to achieve fertilization and seem to stimulate the Leydig cell production of testosterone through the regulation of the steroidogenic acute regulatory protein, a transport protein that regulates cholesterol transfer within the mitochondria, which is the rate-limiting step in the production of steroid hormones. As regards to female fertility, PUFA supplementation mediates a broad range of actions in reproductive processes involving pregnancy establishment, uterine endocrinology, and preterm birth. The perfectly composed follicular environment shapes oocyte quality and thus female fertility. Since both oocytes and embryos are vulnerable to microenvironment changes, nutritional alterations and FA unavailability can lead to their defects. The aim of the present review is to examine the effects of n-3 and n-6 PUFAs on male and female reproductive performances and the correlated endocrine mechanisms.

This is a review of original data concerning new extra- and intracellular regulators of rabbit ovarian functions. Effects of some hormones including leptin, ghrelin, oxytocin, arginine-vasotocin, endothelin (ET-1), gonadotropin releasing hormone (GnRH), adrenocorticotropic hormone (ACTH), growth factors such as insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nuclear peroxisome proliferator-activated receptor gamma (PPARγ), pharmacological regulators of some protein kinases such as protein kinase A (PKA), mitogen-activated protein (MAP) kinase, cell division cycle protein 2 homolog (CDC2 kinase, CDK), tyrosine kinases), and plant molecules (resveratrol, rapamycin) on the functions of ovarian cells (proliferation, apoptosis, secretory activity, expression of some protein kinases) and reproductive end points (blood level of reproductive hormones, ovarian morphology, number of ovulations, embryo yield and quality, number and viability of offspring), and their possible interrelationships and practical application in rabbit breeding are reviewed.

KEY WORDS fasting; LH; estradiol receptor; GnRH receptor; rabbit; pituitaryABSTRACT To investigate the mechanisms by which caloric restriction affects reproductivefunction in female rabbits, we measured, in animals intact or ovariectomized (OVX) estrogen-primed and fed ad libitum or fasted for 48 h, the adenohypophysial expression of estrogenreceptor-alpha (ESR1) and gonadotropin releasing hormone receptor (GnRHR) and the dynamicsecretion of LH following GnRH stimulation. Fasting increased the number of GnRHR-immunoreactive (-IR) cells in intact animals, whereas reduced the density of ESR1-IR cells inOVX rabbits. Estrogen priming decreased thenumber of ESR1-IR cells in fasted and OVX ani-mals. Ovariectomy increased the number of ESR1-IR cells in fed rabbits, but caused an oppositeeffect in both fed and fasted animals treated with estrogen. Fasting down regulated the mRNAlevels for ESR1 and GnRHR. Estrogen-priming reduced the abundance forESR1 mRNA in bothfed and fasted rabbits, and that forGnRHR in fasted rabbits. Ovariectomy halvedESR1 mRNAlevels independently of treatment and feeding condition, whereas increased (P

This research was undertaken to determine the glycoconjugates secreted by the glandular epithelium of the seminal vesicles in the entire and castrated horses using lectin histochemistry in combination with sialidase digestion and deglycosylation pre-treatments. The following lectins were used: Con-A, UEA-I, LTA, WGA, GSA-IB4, SBA, PNA, ECA and DBA. In the entire stallion, the glandular cells expressed the following sugar residues: α-Fuc, internal GlcNAc, terminal β- and α-GalNAc, β-D-Gal-(1–4)-GlcNAc and α-Gal included in O-linked oligosaccharides; β-Gal-(1–3)-GalNAc belonged to both O- and N-oligosaccharides whereas terminal GlcNAc belonged to N-linked glycans. Additionally, α-Gal and β-Gal acted as acceptor sugars for sialic acid moieties. In castrated horses, the glandular epithelium showed a different lectin labelling pattern. In particular, we evidenced internal GlcNAc, α-GalNAc, β-Gal-(1–3)-GalNAc in both O- and N-linked glycoproteins whereas β-GalNAc and β-Gal-(1–3)-GalNAc in O-linked glycoproteins...

To study the role of endothelial factors in luteal function, the dynamic profiles of genes for endothelin 1 (EDN1), its receptor subtypes, EDNRA and EDNRB, and angiotensin converting enzyme (ACE) were examined in corpora lutea (CL) obtained from rabbits on Days 4 and 9 of pseudopregnancy after prostaglandin (PG) F2alpha analogue (alfaprostol) treatment. The cell type distribution of EDN1 in the ovaries and its mechanisms of actions in vitro and in vivo were also studied. Positive immunostaining for EDN1 was localized in the luteal and endothelial cells, in granulosa cells of the follicles, and in the ovarian epithelium. The basal mRNA levels for EDNRA, EDNRB, and ACE were lower (P

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well asin vitroPGE2 and PGF2α synthesis following PGF2α treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P< 0.01) 4- to 10-fold 1.5–6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P< 0.01) COX-2 and PGE2-9-K basal activities, and PGF2α synthesis rate, but higher (P< 0.01) PGE2 production. Independent of luteal stage, PGF2α treatment did not affect COX-1 activity. In day-4 CL, PGF2α induced an increase (P< 0.01) in both COX-2 activity and PGF2α synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2α up-regulated (P< 0.01) both COX-2 and PGE-9-K activities, and PGF2α production, but decreased (P< 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2α challenge and were more marked in day-9 CL. Our data suggest that PGF2α directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2α synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2α in rabbits.

This study examined the effects of food restriction during rabbit pregnancy on hormones and metabolites involved in energy homeostasis and metabolic programming. Pregnant does were assigned to four groups: the control group was fed a standard ration while the others received a restricted amount of food (30% restriction) during early (0–9 days), mid (9–18 days), and late (19–28 days) pregnancy. The pregnancy induced a coordinated range of adaptations to fulfil energy requirements of both mother and foetus, such as hyperleptinaemia and hyperinsulinaemia, reduced insulin sensitivity, increased cortisol and non-esterified fatty acid. Food restriction altered leptin, insulin, T3, non-esterified fatty acids and glucose concentrations depending on the gestational phase in which it was applied. Collectively, present data confirm that the endocrinology of pregnancy and the adaptive responses to energy deficit make the rabbit an ideal model for studying nutritional-related disorders and foetal programming of metabolic disease.

Experiments were devised to characterize the expression of nerve growth factor, beta polypeptide (NGF), and its cognate receptors neurotrophic tyrosine kinase receptor type 1 (NTRK1) and nerve growth factor receptor (NGFR) in rabbit male sex organs, as well as the concentrations of NGF in both seminal and blood plasma of sexually mature male rabbits. Immunoreactivity and gene expression for NGF and cognate receptors were detected in testis, prostate gland and seminal vesicle. The highest levels of NGF and NTRK1 transcripts were found in the prostate, while intermediate expressions were found in the testis. NGFR transcripts were expressed at the same levels in both testis and prostate and were more abundant than in seminal vesicles. The widespread distribution of NGF in all prostate glandular cells, together with its relative high mRNA abundance, confirms that the prostate of rabbits is the main source of this neurotrophin. In conclusion, the present data suggest that the NGF system is involved in the testicular development and spermatogenesis of rabbits and that NGF may act as a potential ovulation-inducing factor being abundantly present in the seminal plasma.

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 microg iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ET(A)/ET(B) receptor antagonist, or cyclooxygenase (COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F2alpha (PGF2alpha). In CL cultured in vitro, ET-1 increased (P/= 0.01) both PGF(2alpha) production and luteal nitric oxide synthase activity but decreased (Por = 0.01) progesterone release. Addition of ET(A) receptor antagonist BQ123 or COX inhibitor blocked the ET-1 luteolytic effects. Positive staining for ET-1 receptors was localized in ovarian blood vessels, granulosa cells of large follicles, and luteal cells. Immunoblot analysis of ET-1 receptor protein revealed a strong band of approximately 48 kDa in d-9 CL. Up to d 6 of pseudopregnancy, ET-1 mRNA abundance in CL was poorly expressed but then increased (Por = 0.01) at d 9 and 13. ET(A)-receptor transcript increased (Por = 0.01) at d 6, remained at the same level up to d 13, and then declined to the lowest (Por = 0.01) levels at d 22. ET(B)-receptor mRNA increased (Por = 0.01) throughout the late-luteal stage from d 13 up to d 18. Our data suggest that the luteolytic action of ET-1 may be a result of PGF2alpha synthesis from both luteal and accessory cells, via the COX pathways.

Angiotensin converting enzyme (ACE) was demonstrated to modulate the production of 17beta-estradiol, progesterone and prostaglandin E2 (PGE2) in frog ovary of Rana esculenta. However, the activity was not mediated by angiotensin II (Ang II). In an attempt to identify the peptide involved in the pathway modulated by ACE, bradykinin, another physiological substrate of ACE, was chosen and incubated in the presence of the membrane suspension purified from the frog ovary homogenate. The hydrolytic products were analysed by reverse-phase high-pressure liquid chromatography (HPLC) analysis and the results showed that bradykinin was metabolized by membrane suspension. The presence of the protease inhibitors in the incubation mixture indicated ACE and neutral endopeptidase as being responsible for the bradykinin hydrolysis. Frog ovary was incubated in vitro in the presence of bradykinin (10 microM), bradykinin receptor antagonist NPC 567 (1 mg mL-1), bradykinin fragment (1-7) (10 microM), ACE (2.5 mU mL-1), captopril (0.1 mM) and lisinopril (0.1 mM). The results showed no modulating activity by bradykinin on ovarian 17beta-estradiol and PGE2 production, thus demonstrating that it was not involved in the ACE-modulated pathway.

The in vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate (DEHP) on the reproductive function of peroxisome proliferator-activated receptor gamma (PPARG) were studied in rabbit corpora lutea (CL) at early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. The rabbits were in vivo treated with DEHP for 15 days before induction of pseudopregnancy. Immunohistochemistry provided evidence for the presence of PPARG, prostaglandin endoperoxide synthase 1 (PTGS1), PTGS2, prostaglandin E2-9-ketoreductase (PGE2-9-K), and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in all the luteal cells during pseudopregnancy. DEHP decreased progesterone plasma levels and CL production in all the luteal stages and PPARG protein and gene expressions in early and mid-CL. DEHP in vivo treatment reduced PTGS2 protein expression at the late stage and that of PGE2-9-K at all the stages, whereas PTGS1 and 3beta-HSD were not affected. In in vitro cultured CL, DEHP alone, the PPARG antagonist T0070907 alone, or DEHP plus T0070907 diminished progesterone production and 3beta-HSD activity and increased PGF2alpha and PTGS2 in early and mid-CL, whereas DEHP plus the PPARG agonist 15d-PGJ2 did not affect these hormones and enzymes. All the in vitro treatments did not affect PGE2 secretion as well as PTGS1 and PGE2-9-K enzymatic activities in all the luteal stages. These results provided evidence that DEHP favors functional luteolysis of pseudopregnant rabbit CL, with a mechanism that seems to involve PPARG expression down-regulation, an increase of PTGS2 activity and prostaglandin F2alpha secretion, 3beta-HSD down-regulation, and decrease in progesterone.

Systemic and local infections and inflammations are known to cause infertility in humans and animals. However, the mechanisms by which infection/inflammation induces infertility are only partially known. The objectives of this study were: (i) to provide models of systemic (acute) and local (sub-acute) inflammation by intra-peritoneal injection or intra-cervical deposition of lipopolysaccharide (LPS) in the rabbit and (ii) to assess their effects on uterine tissues and sperm transport in the genital tract of rabbit does. Intra-peritoneal administration of different doses of LPS induced systemic effects such as fever, anorexia and changes in white blood cells (WBC) count. In our study, LPS inoculation (100μg/kg) produced an inflammation-like status that lasted for about 3 days, with minimal distress for the animals. Intra-peritoneal administration of LPS 60h before artificial insemination induced a rapid increase of IL-1β concentrations. The intra-cervical inoculation of LPS did not show any systemic effects, as confirmed by the lack of changes in body temperature, feed intake and WBC count. Histological examination of uterine tissues showed an endometritis-like inflammation status in LPS-treated does, more severe in those inoculated intra-cervically. The number of spermatozoa recovered from uterine horns and oviducts of intra-cervically treated does was less than that retrieved from intra-peritoneally treated animals and controls. These results suggest (i) that sub-acute or acute inflammation may cause infertility by compromising the uterine environment and/or impairing sperm transport and (ii) that the LPS-induced -infection/inflammation experimental model is useful for studying the mechanisms involved in reproductive dysfunctions in the rabbit.

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

The possible role of Substance P (SP) was studied in the modulation of basal and gonadotopin-releasing hormone (GnRH)-induced gonadotropin secretion in the urodele crested newt, Triturus carnifex. During prereproduction, reproduction (noncourtship and courtship), refractory, recovery and aestivation, male and female pituitaries were incubated with medium-alone, GnRH, SP, GnRH receptor antagonist (antide), and SP receptor antagonist (L-703606). Since antisera raised against gonadotropins are not available for this species, we measured these hormones indirectly through their effects on the secretion of testicular androgens and ovarian progesterone from gonads superfused with the preincubated pituitaries. Pituitaries of both sexes preincubated with medium-alone, GnRH, GnRH plus L-703606, and GnRH plus SP plus L-703606 increased steroid secretion during prereproduction, noncourtship, courtship, and recovery; the increase induced by the pituitaries incubated with medium-alone was lower during prereproduction, noncourtship, and recovery. Pituitaries preincubated with SP, GnRH plus SP, GnRH plus SP plus antide, and SP plus antide did not change basal steroid secretion in any of the reproductive phases considered. Antide, L-703606, GnRH plus antide, GnRH plus SP plus antide plus L-703606, SP plus L-703606, and antide plus L-703606 experimental groups showed the same results as those with medium-alone. These results suggest that SP downregulates gonadotropin release in both Triturus carnifex sexes. In addition, an antagonist role, through receptor-independent mechanisms, exists between GnRH (upregulation) and SP (downregulation) in the modulation of pituitary.

The role of nitric oxide (NO) synthase, prostaglandin E2-9-ketoreductase, and aromatase brain activities in regulating frog amplexus was assessed in the water frog (Rana esculenta). Plasma concentrations of testosterone were higher, and concentrations of 17beta-oestradiol lower, in amplexing males than in unamplexing males; while concentrations of testosterone and PGE2 were lower, and those of 17beta-oestradiol and PGF2alpha higher, in amplexing females compared to unamplexing females. Hormone release rescued from frog brains in vitro mirrored plasma hormone measures. Brain aromatase activity was lower in amplexing males; NO synthase was lower and PGE2-9-ketoreductase and aromatase were higher in amplexing females. In male brains, PGE2-9-ketoreductase inhibitor decreased PGF2alpha release and increased that of PGE2; aromatase inhibitor decreased 17beta-oestradiol and increased testosterone release. In female brains, NO donor and PGE2-9-ketoreductase inhibitor increased testosterone and PGE2 release and decreased that of 17beta-oestradiol and PGF2alpha; NO synthase inhibitor decreased testosterone release and PGE2 and increased 17beta-oestradiol and PGF2alpha release; PGF2alpha decreased testosterone release and increased 17beta-oestradiol release; aromatase inhibitor decreased 17beta-oestradiol release and increased testosterone release. In female brains, NO donor and PGE2-9-ketoreductase inhibitor decreased PGE2-9-ketoreductase and aromatase activities; PGF2alpha increased aromatase activity; NO synthase inhibitor increased PGE2-9-ketoreductase and aromatase activity. The data suggest that, in amplexing female brains, external and/or internal stimuli inhibit NO synthase, decreasing NO and activating PGE2-9-ketoreductase; in turn, PGF2alpha increases aromatase activity and 17beta-oestradiol release; while, in amplexing male brains, stimuli inhibit aromatase activity, thereby increasing testosterone production.