Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

The abundance and localization of heat shock proteins (HSP)-60, -70, and -90 in the oviductal ampulla of hamadryas baboon (Papio hamadryas) during the menstrual cycle.

The presence of HSPs in female reproductive and their relationship with the steroid hormone fluctuation have been reported in several mammals but not in non-human primates. The present research dealt with the oviductal expression and localization of the more studied HSPs (60, 70, and 90) as well as the morphological changes in the Hamadryas baboon (Papio hamadryas) during the follicular, preovulatory, and luteal phases of the menstrual cycle. Therefore, western blots, histomorphological, and immunohistochemical analyses were carried out. The results of western blot analysis displayed the lowest HSP expression in the luteal phase. The histomorphology showed that the mucosal epithelium consisted of undifferentiated cuboidal cells in follicular and luteal phases and well-distinguishable columnar ciliated and non-ciliated cells during the preovulatory phase. Immunohistochemistry evidenced that the mucosal epithelium contained cytoplasmic and nuclear HSP60, 70, and 90 immunostaining in the follicular and luteal phases. During the preovulatory phase, the non-ciliated cells showed: (i) cytoplasmic HSP60; (ii) nuclear and cytoplasmic HSP90. Ciliated cells showed cytoplasmic and ciliary HSP70 and ciliary HSP90. The stromal cells and myocytes of muscular layer displayed a decreased cytoplasmic HSP60 in the preovulatory phase and nuclear and low cytoplasmic HSP70 throughout the menstrual cycle. Nuclear HSP90 decreased in ampulla stromal cells and the follicular phase myocytes. These findings indicate that the expression pattern of HSP60,70, and 90 is related to the morphofunctional features of the baboon oviductal ampulla during the menstrual cycle and could represent a referent point for further studies in the oviduct of Primates.

Modification of Morphology and Glycan Pattern of the Oviductal Epithelium of Baboon Papio hamadryas during the Menstrual Cycle.

The mammalian oviduct is a highly specialized structure where fertilization and early embryonic development occur. Its mucosal epithelium is involved in maintaining and modulating a dynamic intraluminal fluid. The oviductal epithelium consists of ciliated and non-ciliated (secretory) cells whose differentiation and activity are sex hormone-dependent. In this study, we investigated for the first time both the morphology and the glycan composition of baboon oviductal epithelium during the menstrual cycle. Oviducts were laparoscopically removed from 14 healthy adult female Papio hamadryas whose menstrual cycle phase was assessed based on the sex hormone levels and the vaginal cytology features. Histological investigations were carried out on fimbriae, infundibulum, ampulla, and isthmus separately fixed in 4% (v/v) paraformaldehyde, embedded in paraffin wax, and stained with hematoxylin-eosin for morphological analyses and using a panel of nine fluorescent lectins for glycoconjugate characterization. The histomorphological analysis revealed that in the entire oviduct (i) the ciliated and non-ciliated cells were indistinguishable during the follicular and luteal phases, whereas they were highly differentiated during the preovulatory phase when the non-ciliated cells exhibited apical protrusions, (ii) the epithelium height was significantly higher in the preovulatory phase compared to other menstrual phases, and (iii) the number of ciliated cells significantly (p ≤ 0.05) increased from the fimbriae to the infundibulum and progressively reduced in the other oviductal segments with the lower presence of ciliated cells in the isthmus. The glycan characterization revealed a complex and region-specific composition during the different phases of the menstrual cycle. It can be summarized as follows: (i) high-mannosylated N-linked glycans (Con A reactivity) were present throughout the oviductal epithelium during the entire menstrual cycle and characteristically in the apical protrusions of non-ciliated cells of the ampulla during the preovulatory phase; (ii) sialoglycans with α2,3-linked sialic acids (MAL II binding) were expressed along the entire oviductal surface only during the preovulatory phase, whereas α2,6-linked ones (SNA affinity) were also detected in the surface of the luteal phase, although during the preovulatory phase they were characteristically found in the glycocalyx of the isthmus cilia, and O-linked sialoglycans with sialic acids linked to Galßl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA) were found in the entire oviductal surface during all phases of the menstrual cycle; (iii) GalNAc terminating O-linked glycans (HPA staining) were mainly expressed in the entire oviducts of the luteal and preovulatory phases, and characteristically in the apical protrusions of the isthmus non-ciliated cells of the preovulatory phase; and (iv) fucosylated glycans with α1,2-linked fucose (LTA reactivity) occurred in the apical surface of fimbriae during the luteal phase, whereas α1,3/4-linked fucose (UEA I binders) were present in the apical protrusions of the ampulla non-ciliated cells and in the apical surface of isthmus during the preovulatory phase as well as in the isthmus apical surface of follicular-phase oviducts. These results demonstrate for the first time that morphological and glycan changes occur in the baboon oviductal epithelium during the menstrual cycle. Particularly, the sex hormone fluctuation affects the glycan pattern in a region-specific manner, probably related to the function of the oviductal segments. The findings add new data concerning baboons which, due to their anatomical similarity to humans, make an excellent model for female reproduction studies.

Comparison of Two Radiofrequency Vessel-Sealing Device for Laparoscopic Ovariectomy in African Lionesses (Panthera Leo).

To evaluate two vessel-sealing devices with different jaw lengths for dissecting ovaries in adult obese African lionesses undergoing laparoscopic ovariectomy. Twelve lionesses (n = 12) were recruited. The surgical procedures were performed through three portals, with a retractor platform positioned at the umbilical port and cannulas placed 3-4 cm cranial and caudal to the device at the level of the midline. Ovariectomy was performed using a vessel-sealing device according to a randomization list. We evaluated the surgery time the intra- and postoperative complications. The total surgery time was 49.3 min (range 40-61 min) in the Atlas group and significantly lower in the Caiman group (mean 31.8 min, range 26-51 min). The installation phase was similar between the groups. The ovariectomy time was significantly lower in the Caiman group (mean 7.8 min, range 4-11 min) than in the Atlas group (mean 20 min, range 16-30 min). Controlled bleeding was observed at the tip of the uterine horn in two cases in the Atlas group. No other complications were noted. The results of our study confirmed the significant advantages of employing the Caiman 12 vessel-sealing device in comparison with the LigaSure Atlas in terms of the time needed to complete ovariectomy, although both instruments could be considered safe. The use of the Caiman 12 is recommended when performing laparoscopic ovariectomies in adults with obesity.

The role of bicarbonate in the modulation of capacitation, spontaneous acrosome reaction and motility of equine fresh and frozen spermatozoa.

In this study, we defined the composition of the culture medium that yield a significant percentage of alive and functional equine spermatozoa during enough time before artificial insemination. The effects of sodium bicarbonate were analyzed in fresh and frozen semen in respect to sperm viability, capacitation, spontaneous acrosome reaction and several kinetic parameters such as total motility, progressive motility, VCL, VSL, ALH, BCF, LIN. Moreover, employing Bayk-6844 and Nifedipine, the involvement of L-type voltage-gated calcium channels in the modulation of intracellular calcium concentrations was investigated. Results evidenced an immediate effect of sodium bicarbonate in reducing fresh sperm viability and in increasing capacitation and spontaneous acrosome reaction of fresh and frozen spermatozoa. Furthermore, it affected total and progressive motility of fresh and frozen semen. Because of the sudden effects of the compound, we think that a buffer lacking sodium bicarbonate is more suitable to preserve the viability and the functional state of equine spermatozoa required to undergo at the right time those modifications necessary for fertilization. We also demonstrated that intracellular calcium modifications induced by Bayk-8644 and Nifedipine are not involved in signals related to vitality, capacitation, spontaneous acrosome reaction and motility.

Rabbit metabolic response and selected meat quality traits: Evaluation of dietary PLX® 23 and LycoBeads® feed supplement.

Effects of dietary supplementation of verbascoside and lycopene, alone and in combination, on some blood parameters, plasma oxidative status and meat quality traits in intensively reared rabbits were investigated. The test lasted 60 days and was conducted on 200 weaned-rabbits, divided into four groups of 50 animals each (5 animals each cage × 10 repetitions). The control group (CON) received a fattening feed without any feed supplements, whereas the experimental groups received the following: the first, an integration in 22 g of PLX® 23 (Lippia citriodora extract; VB group) per ton of feed; the second, an integration in 100 g of LycoBeads® (Solanum lycopersicum extract; LIC group) per ton of feed; and the third, an integration of both feed supplements in combination at the same doses (LIC+VB group). The dietary supplementation with Lippia citriodora and Solanum lycopersicum resulted in an improvement of the blood lipid profile, oxidative plasma markers, and hepatic and renal activity of treated growing rabbits. Feed additives also produced meat with a lower content in SFA and an increased PUFA content, and inhibited the lipid oxidation improving the oxidative stability of rabbit meat. The dietary supplementation, even reporting no effects on productive parameters and carcass yield, showed an improvement on several blood parameters, as indicator of animal welfare, and on quality and healthy meat markers.

Dietary effect of lemon verbena extract on selected blood parameters and on plasma oxidative profile in Avelignese horses.

The effect of Lippia citriodora extract on selected blood parameters and on plasma oxidative markers in Avelignese horses was evaluated. Twenty-four horses were divided into three groups, consisting of eight animals each. Results of two experimental groups, 0.5 mg of verbascoside per kg of metabolic body weight (bw0.75 ) in the low-dose group (LVB) and 1.0 mg of verbascoside per kg of metabolic body weight (bw0.75 ) in the high-dose group (HVB), were compared to the control group (CON). Groups fed L. citriodora extract (HVB and LVB) showed a significant decrease in triglycerides, total cholesterol and LDL cholesterol (p < .01), bilirubin, and transaminases (p < .05), and an increase in HDL cholesterol (p < .01) compared to the CON group. Oxidative status was improved due to significant decrease in plasma concentration of ROMs and TBARS (p < .01) and increase in levels of vitamin A and vitamin E (p < .01). Based on obtained results, it is assumed that dietary supplementation with L. citriodora extract might find a useful application in horse feeding, with positive impact observed in blood parameters and plasma oxidative markers, with beneficial effects on the physiological welfare of livestock animals.

Long-term shedding of Canine alphaherpesvirus 1 in naturally infected newborn pups.

The long-term shedding of Canine alphaherpesvirus 1 (CaHV-1) by neonatal pups with natural infection is reported. The pups belonged to a litter of 11 pointers of a breeding kennel in southern Italy, 9 of which developed a fatal form of systemic infection, as resulted by the detection of CaHV-1 in internal organs (kidney, liver, lung and brain) of one of this dogs and in the vaginal swab of their mother. The two remaining animals displayed a milder form of disease, with one pup showing ocular involvement, and underwent a progressive recovery. These pups were monitored from 11 to 36  days of age, showing a long-term shedding of the virus through the nasal and ocular secretions and the faeces. CaHV-1 shedding, as assessed by means of a specific and sensitive real-time PCR assay, occurred mainly through the nasal secretions, although the pup displaying ocular disease shed the virus at high titres and for a long period even in the ocular secretions.

Follicular development in pregnant cows after the administration of equine chorionic gonadotropin (eCG): a new insight.

The follicular development in the cow occurs in a wave-like pattern, and it takes place also during pregnancy. In the cow, Equine Chorionic Gonadotropin (eCG) is used for superovulation, but a decrease in total fertility has been reported, likely because of its immunogenic properties in species other than equine. In this regard, immune response has been implicated in follicular growth, ovulation, and placental development. So, aims of our study are to test the safety of eCG administered during pregnancy and characterize the ovarian activity, the quality of oocytes, the hormonal status, and interleukin levels in eCG-treated pregnant cows.

Apoptosis in canine corpus luteum during spontaneous and prostaglandin-induced luteal regression.

Spontaneous luteal regression and prostaglandin-induced luteolysis in bitches were evaluated by measuring the apoptotic index for DNA fragmentation and the relative level of Bax gene expression in ovaries removed from nine untreated nonpregnant bitches at selected times during diestrus and in nine pregnant bitches after 1 day of administering abortive doses of a PGF-analog gel formulation given intravaginally at selected times during gestation. Nonpregnant diestrus was divided into three periods (early, mid and late) based on vaginal cytology and plasma progesterone concentration. Pregnant bitches were treated with a PGF-analog gel at corresponding stages of pregnancy (early, mid and late) and evaluated by ultrasound. Another eight pregnant bitches were similarly studied and serum progesterone concentrations were determined after 1, 2, 3 or 4 days of PGF-analog gel. Corpora lutea obtained by ovariohysterectomy were analyzed for apoptotic internucleosomal DNA fragmentation relative to that in a control cell line (U937), using an apoptotic DNA ladder kit and gel electrophoresis and for relative expression of the pro-apoptotic Bax gene by RT-PCR and electrophoresis. In nonpregnant bitches, the DNA fragmentation apoptotic index was greater in late than in early diestrus (P < 0.01). The index after 1 day of PGF-analog gel was higher in early pregnant bitches than in early diestrus bitches (P < 0.05); it was highest in midpregnancy (P < 0.05). The degree of apoptosis was related to the number of times PGF-analog gel was administered. Bax mRNA was detected in the corpus luteum (CL) and Bax expression increased from early to middiestrus in nonpregnant subjects (P < 0.05). Potential elevation in Bax due to PGF-analog gel treatment in pregnancy was only significant in relation to normal diestrus during early pregnancy (P < 0.01). In conclusion, we inferred that the effects of endogenous or exogenous prostaglandin on CL life span in bitches involved increases in apoptotic activity and that increased apoptosis was implicated in normal luteal regression in nonpregnant bitches.