GnRH analogue triptorelin acetate induces ovulation in jennies during oestrus.

Most of the northern hemisphere donkey breeds are faced with the risk of extinction, thus donkey reproduction is considered an emerging branch of theriogenology, and the management of artificial insemination and induction of ovulation is a crucial point in setting up preservation protocols. For four consecutive cycles, six jennies' ovarian activity was routinely monitored; an ultrasound examination was performed daily from the evidence of a follicle diameter ≥27 mm until ovulation. Upon reaching a follicular diameter ≥32 ± 2 mm (Hour 0), oestrous jennies were treated alternatively with 0.1 mg triptorelin acetate, sc, (TRIP), 0.4 mg/sc of buserelin acetate (BUS) or saline, sc, (CTRL) and examined ultrasonographically at Hours 14, 24, 38, 42, 48, 62 and every 24 h until ovulation. Induction of ovulation was considered successful if ovulation occurred from 24 to 48 h after treatment. 11/12 cycles resulted in ovulation for TRIP and 12/12 for BUS and CTRL groups, respectively. Mean ± SD ovulation time after treatment was 37.3 ± 8.3, 47.1 ± 21.0 and 66.8 ± 25.9 h for BUS, TRIP and CTRL, respectively (p = .0032). Ovulation rates between h24 and h48 were 10/12 (83.3%) for both TRIP/BUS and 2/12 (16.7%) for CTRL, respectively (p = .003). Buserelin and triptorelin-treated jennies had a 25 times higher probability to ovulate between Hours 24 and 48 than controls (p = .003), while there were no jenny and cycle effects on the ovulatory rate. The results of this study show how triptorelin successfully induced ovulation in jennies, like other GnRH analogues previously evaluated.

Convergently evolved placental villi show multiscale structural adaptations to differential placental invasiveness.

Despite having a single evolutionary origin and conserved function, the mammalian placenta exhibits radical structural diversity. The evolutionary drivers and functional consequences of placental structural diversity are poorly understood. Humans and equids both display treelike placental villi, however these villi evolved independently and exhibit starkly different levels of invasiveness into maternal tissue (i.e. the number of maternal tissue layers between placental tissue and maternal blood). The villi in these species therefore serve as a compelling evolutionary case study to explore whether placentas have developed structural adaptations to respond to the challenge of reduced nutrient availability in less invasive placentas. Here, we use three-dimensional X-ray microfocus computed tomography and electron microscopy to quantitatively evaluate key structures involved in exchange in human and equid placental villi. We find that equid villi have a higher surface area to volume ratio and deeper trophoblastic vessel indentation than human villi. Using illustrative computational models, we propose that these structural adaptations have evolved in equids to boost nutrient transfer to compensate for reduced invasiveness into maternal tissue. We discuss these findings in relation to the 'maternal-fetal conflict hypothesis' of placental evolution.

Endometrial expression of antimicrobial peptides as markers of subclinical endometritis in mares.

BACKGROUND: Endometritis is a major cause of subfertility in mares. Multiparous old mares are more susceptible to developing endometritis given that ageing is associated with an altered immune response and with inadequate physiological uterine clearance after breeding, which can lead to degenerative changes in the endometrium. Molecules such as antimicrobial peptides (AMPs) have been proposed as endometritis markers in the equine species. STUDY DESIGN: Cross-sectional. OBJECTIVES: To investigate the endometrial expression of defensin-beta 4B (DEFB4B), lysozyme (LYZ) and secretory leukocyte peptidase inhibitor (SLPI) genes in mares either affected or not by subclinical endometritis, due to the role of these AMPs in the immune response to bacteria and inflammatory reactions. METHODS: Endometrial biopsy for histopathological and gene expression examinations was performed on 26 mares. The inclusion criteria for the normal mare group (NM, N = 7) were 2-4 years of age, maiden status, no clinical signs of endometritis and a uterine biopsy score of I, while for mares affected by subclinical endometritis (EM, N = 19) the inclusion criteria were 10-22 years of age, barren status for 1-3 years, no clinical signs of endometritis and a uterine biopsy score between IIA and III. RESULTS: A significantly higher expression of LYZ (NM: 0.76 [1.84-0.37] vs. EM: 2.78 [5.53-1.44], p = 0.0255) and DEFB4B (NM: 0.06 [0.11-0.01] vs. EM: 0.15 [0.99-0.08], p = 0.0457) genes was found in endometritis mares versus normal mares. Statistically significant moderate positive correlations were found between the level of expression of LYZ gene and both the age (r = 0.4071, p = 0.039) and the biopsy grade (r = 0.4831, p = 0.0124) of the mares. MAIN LIMITATIONS: The study investigated a limited number of genes and mares, and the presence/location of the proteins coded by these genes was not confirmed within the endometrium by IHC. CONCLUSIONS: If the results of this study are confirmed, LYZ and DEFB4B genes can be used as markers to identify mares that are affected by subclinical endometritis.

Autonomic nervous system balance in parturient mares: Spontaneous vs induced delivery.

Delivery is not easily predictable in horses and the consequences of dystocia can be serious for both the mare and foal. An induction protocol with low doses of oxytocin has been reported as a safe procedure. This study investigates the effect of induced delivery on at-term mares' sympathetic-vagal balance. Fourteen mares were included and divided into two groups, one subjected to spontaneous delivery (SD), and one to induced delivery (ID). In both groups, an ECG was recorded using an elastic belt with integrated smart textile electrodes. The recording started before the delivery (Basal), continued close to delivery (Pre-delivery) and during delivery (Delivery), and ended after parturition (Placental expulsion). From the ECGs, Heart Rate Variability (HRV) parameters relating to time and frequency domains and non-linear analysis were extrapolated. The HRV analysis was performed both within the same group (IntraGA) and between the two groups (InterGA). In the present study, spontaneous and induced delivery did not appear to differ in autonomic nervous system functioning. In IntraGA analysis, both for SD and ID mares, delivery and placental expulsion periods were parasympathetic dominated since vagal-related HRV parameters increased. Moreover, no differences were found in InterGA comparison between SD and ID mares, except for the pre-delivery period of ID mares, during which both branches of the autonomic nervous system were activated. These results are in line with the literature on parasympathetic dominance during parturition and no change in Heart Rate Variability following exogenous oxytocin administration in parturient mares.

Interspecific and Intraspecific Artificial Insemination in Domestic Equids.

Horses and donkeys differ phenotypically and karyotypically, although they can interbreed freely. Eight Standardbred mares and nine Amiata donkey jennies were included in the study. Semen was collected from two horses and two donkey stallions of proven fertility. A first pregnancy diagnosis was performed on day 10 after ovulation and repeated every day until embryo detection or until day 16. Irrespectively of the sire species, pregnancy rates in horse females (20/30, 66.7%) were significantly higher than in donkey females (19/70, 27.1%) (p < 0.05), while horse and donkey males did not affect pregnancy rates. Comparing overall intraspecific and interspecific AI, pregnancy rates were 25/37 (67.6%) and 14/63 (22.2%), respectively (p = 0.0001). The lowest pregnancy rate was obtained when inseminating jennies with horse stallion semen (8/49, 16.3%). No statistical differences were found when comparing embryo diameters, day at first pregnancy diagnosis, or in vitro embryo morphological quality among groups. In this study, much poorer results were obtained with jennies than with mares. Interspecific AI resulted in lower pregnancy rates than intraspecific Al, and AI to produce hinny hybrids resulted in the lowest pregnancy rate. Further studies are required to better understand the mechanism involved in such different outcomes in relation to intra- and interspecific breeding in domestic equids.

The Kisspeptin analogue C6 induces ovulation in jennies.

Kisspeptins (KPs) are the most potent stimulating neurotransmitters of GnRH release, and consequently KP administration triggers LH and/or FSH release. In small ruminants, KP or its analogs induced an LH surge followed by ovulation in both cyclic and acyclic animals, while in the mare KP only increased LH plasma levels but failed to induce ovulation. This study in jennies compares the endocrinological effects, ovulatory and pregnancy rates of the KP analog C6 and the GnRH analog buserelin acetate. The ovarian activity of nine Amiata jennies was monitored daily by transrectal ultrasound for three complete estrous cycles. Jennies in estrus were assigned, to one of three treatment groups: 50 nmol of the KP analog C6 (injected twice, 24 h apart, C6 group); 0.4 mg buserelin acetate (injected once, Bu group); and 2 mL of saline (injected once, CTRL group). Blood samples were collected at Day-1 (-24 h) Day0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48 and every 24 h until ovulation. Jennies were inseminated once at h24 with fresh extended semen from a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. On days 5, 10, and 14 after ovulation, for every CL the cross-sectional area (CSA) and the vascularized area (VA) were recorded by color doppler ultrasound and measured. Significantly higher plasma LH levels were found after induction between the Bu and CTRL groups at h6 and h8 (P < 0.05), while tendentially higher differences were found between the Bu/C6 groups and CTRL at h10. Five/9, 4/9, and 2/9 jennies ovulated between 24 and 48 h after induction from the Bu, C6, and CTRL groups respectively, (P > 0.05). Correlations between corpora lutea CSA and VA with serum progesterone concentration were r = 0.31, P = 0.01, r = 0.38, P = 0.01, respectively. Pregnancy rates after artificial insemination did not differ among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P > 0.05). Ovulation rates after C6 treatment were comparable to that of Bu, although not different from the CTRL. Pregnancy rates were comparable to the literature in terms of fresh extended donkey semen in every group. This study suggests that stimulation of the Kp system in jennies, in contrast to findings observed in mares, induces ovulation. Further studies using higher doses and/or more animals are needed to better characterize the efficacy of C6 in jennies.

Deep-horn Artificial Insemination With Frozen Thawed Semen After re-extension in Autologous Seminal Plasma May Improve Pregnancy Rates in Jennies.

In the literature, the very low pregnancy rates after artificial insemination (AI) with frozen semen in donkeys were improved in one study after re-extension of the frozen-thawed semen in autologous seminal plasma. The aims of our study were (1) to describe in vitro post-thaw parameters of donkey jackass semen after re-extension in seminal plasma (SP) or in INRA96 and (2) to compare pregnancy rates in jennies bred with frozen-thawed semen using two different AI protocols. Semen collected from two Amiata donkey stallions, known to be fertile, was frozen in INRA96 supplemented with 2% egg yolk, and 2.2% glycerol. From the same jacks, seminal plasma was filtered, and stored at -20°C. According to the in vitro analysis, the post-thaw re-extension in SP some kinematic parameters decreased more over time. Two different AI protocols were tested: inseminating deeply into the uterine horn (DHAI) with or without thawed semen re-extension in SP. Higher pregnancy rates were obtained with re-extension in SP than when AIs were performed without SP. Further studies are needed to confirm the advantage of re-extension in autologous SP of donkey frozen-thawed semen before AI in jennies.

Follicular dynamics during the non-reproductive season in Miranda jennies.

Jennies' follicular wave patterns have not yet been addressed during the non-breeding and transition seasons in anoestrous jennies. Twelve non-pregnant females of the Miranda donkey breed were followed to describe follicular wave characteristics during the non-reproductive season and determine the anoestrous effect in follicular wave patterns. Five jennies enrolled in this study experienced anoestrus during the non-breeding season, but all retained the continuous emergence of follicular waves. The average duration of the waves from emergence to peak was 11.2 ± 0.021 days (3-29 days). The duration of the different type of waves was 9.91 ± 0.034 days for minor waves,12.5 ± 0.232 days for major secondary waves and 12.5 ± 0.057 for major primary waves. The major waves were significantly longer than the minor waves (p < .001). Older jennies presented longer waves (p = .021). In the jennies presenting anoestrus, the wave duration during anoestrus (11.2 ± 0.125 days, n = 31) was not different from the waves detected in the preceding and subsequent ovulatory cycles (11.3 ± 0.084 days, n = 43) (p = .978). The number of follicular waves emerging in each ovulatory cycle (n = 59) was 2.36 ± 0.011, varied from 1 to 4. Only in a small proportion of cycles, one wave (0.8%) was recorded, with 41 cycles (67.2%) presenting two waves; fourteen cycles presenting three waves (24.6%); and three cycles (6.6%) showing 4 waves. It was concluded that follicular waves do occur in anoestrous jennies during the non-breeding season with two-wave cycles being common.

Variables Affecting Veterinary Students' Ability to Accurately Interpret Ovulation in Live Mare Palpation.

In a veterinary medicine curriculum, students' hands-on practice is essential but is still considered one of the major deficiencies in veterinary schools in Europe. After theoretical and basic practical training, students, under the control of experienced veterinarians (supervisors), monitored the reproductive cycle of embryo recipients by transrectal palpation and ultrasound. To evaluate the skills of students, the question "Has she ovulated?" was posed when a dominant follicle ≥ 35 mm was recorded in the previous day's examination and a score of 1 or 0 was assigned in the case of a correct or incorrect answer (test palpation), respectively. Study 1 involved the retrospective evaluation of 3,509 test palpation records of 43 students (31 females, 12 males) and showed a statistically significant positive correlation between the number of test palpations performed and the proportion of correct answers. There was a statistically significant effect of the number of test palpations performed by each student, their gender, and the season on the correct answers. When performing > 50 test palpations, a statistical difference between gender was observed (p < .05). Study 2 involved the prospective evaluation of 687 records on 52 standardbred or thoroughbred recipient mares collected from nine right-handed female students. The different mares, breed, occurrence of ovulation on the left or right ovary, and the presence of one or more large follicle(s) per ovary had no effect on the correct answers (p > .05). Individual students' performances were statistically different (p < .05), ranging from 60% to 92%.

hCG is more effective than the GnRH agonist buserelin for inducing the first ovulation of the breeding season in mares.

BACKGROUND: Human Chorionic Gonadotropin (hCG) and Gonadotropin Releasing Hormone agonists (GnRHa) are routinely used to induce ovulation in mares. However, GnRHa efficacy in transitional mares has been suggested to be low. OBJECTIVES: The aims of this study were as follows: (a) to compare the efficacy of hCG and GnRHa in inducing the first ovulation of the breeding season and (b) to evaluate the correlation between ovulatory response, uterine oedema and teasing score at the time of treatment during the early or late transitional phase. STUDY DESIGN: Randomised controlled superiority trial. METHODS: Mares in winter anoestrus were treated with sulpiride when at least two follicles reached a diameter of 25 mm. The day after the follicle reached 35 mm in diameter, mares in oestrus were treated with GnRHa buserelin (N = 29) or hCG (N = 33) and checked daily for ovulation. RESULTS: More mares (30/33, 90.1%) ovulated when the first ovulation after winter anoestrus was induced with hCG, than with GnRHa, (11/29, 38.0%) (P = .0001). Ovulation rate was lower in mares that did not show uterine oedema and full acceptance of the teaser stallion for at least three days before the treatment (32/41, 78% vs 9/21, 42.9%) P = .01. MAIN LIMITATIONS: Plasma LH and oestrogen concentrations were not performed. CONCLUSIONS: These results demonstrate that hCG was more effective than GnRHa for inducing ovulation in the first cycle after winter anoestrus. Uterine oedema and behavioural signs of oestrus, for at least three days before the treatment, were predictors for a positive response to ovulation induction.

Recipients' pregnancy rate was affected by season but not by the temperature-humidity index (THI) in an equine commercial ET programme in Southern Europe.

This study retrospectively analysed the effects of different environmental factors such as the photoperiod, temperature/humidity index of 19 years of a commercial embryo transfer programme performed in central Italy. From 340 donors of different breeds and aged between 2 and 25 years, 576 embryos of an excellent or good quality were obtained by uterine flushing on days 7-8 after ovulation. The embryos were transferred to 259 recipients, aged between 2 and 16 years, and belonging to different breeds. Both the donors and the recipients were employed for several cycles/years. At the time of transfer, the recipients were in diestrus, from day 4 to day 8 after ovulation, and were classified as excellent or good based on clinical examination findings. Out of the total number of embryos transferred, 437 pregnancies were obtained at 14 days (75.9%), with 48/437 (10.1%) embryonic losses at 40 days; with no significant differences among years. Significant differences (p < .05) were observed between 14-day pregnancy rates for transfers performed during the spring transition and breeding seasons (77.1%) compared to those performed during the autumn transitional season (57.9%). No effect of temperature/humidity index was found on pregnancy rates or pregnancy losses after the transfer to the recipients (p > .05). Embryo transfer during autumn was less successful than the transfers performed during the spring transition and the breeding season.

Hastening Time to Ejaculation in Donkey Jacks Treated with the PGF2α Analog, Cloprostenol Sodium.

Due to the long courtship needed to attain excitation and erection, donkey semen collection can take up to 90 min. ProstaglandinF2α (PGF2α) has been reported to hasten the onset of erection and ejaculation in domesticated mammals, presumably by inducing smooth muscle contractions in the internal genitalia. However, while it has been anecdotally used in donkeys, it has yet to be critically evaluated. This study aimed to compare behavioral and semen parameters in Catalan, Balearic, Amiata, and Miranda jacks treated with the PGF2α analogue cloprostenol sodium immediately prior to exposure to an estrus jenny. Nineteen donkeys were assigned in a crossover design to receive cloprostenol sodium (125µg, i.m.; n = 53 collections) or saline (1 mL, i.m.; n = 53 collections). There were no differences for erection (52/53 vs. 52/53) or ejaculation (52/53 vs. 48/53) for collection attempts assigned to saline or cloprostenol sodium, respectively. Cloprostenol sodium significantly hastened treatment-to-erection and treatment-to-ejaculation times from 12.0 ± 1.6 to 6.0 ± 1.6 min and from 14.0 ± 1.4 to 9.6 ± 1.4 min, respectively. Significant effects of breed and age were observed in behavioral and parameters, but there were no effects of cloprostenol sodium administration on semen parameters. In conclusion, cloprostenol sodium administration immediately prior to semen collection hastened time to collect semen in donkeys with no detrimental effects on semen quality and can be used by practitioners to circumvent long delays in donkey semen collection.

One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation.

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

Peripartum findings and blood gas analysis in newborn foals born after spontaneous or induced parturition.

Induction of parturition in horses is still not well accepted due to the potential peripartum complications for mares and newborn foals. We assessed differences after spontaneous and induced parturition with low doses of oxytocin (OX) in 1) incidence of peripartum complications in mares; 2) viability, behavioral, physical, and venous blood gas analyses in foals. In this study 61 mares were included; 45/61 were enrolled in the spontaneous foaling group (SF) and 16/61 in the induced foaling group (IF). In the IF group, when the calcium in mammary secretion reached concentrations of ≥250 ppm, mares received a single injection of 2.5 IU of oxytocin IV once a day until foaling. Mares' breed, age, parity, gestational and stage II length, and peripartum complications were recorded. Foal maturity, vital (Apgar score), behavioral and physical parameters were assessed at birth, and the foal clinical condition was monitored for one week. A jugular venous blood sample was collected at birth for blood gas analysis, acid-base status, and lactate assessment. The median gestational length was within the reference interval in all the mares included and did not differ between the two groups. No statistical differences in the II stage length nor in incidence of peripartum complications were observed between the two groups. All the foals were born alive and showed no signs of prematurity/dysmaturity. No statistical differences were found in foal viability between the two groups. Time to stand and nurse from the mare, and body temperature were significantly higher in the IF compared to the SF group. Venous blood pH, SO2% and BE were lower, while pCO2 and lactate were higher in the IF than in the SF group. All the foals in both groups remained clinically healthy during the observation period. In conclusion, at term induction of parturition with a low dose of oxytocin does not have adverse effects on peripartum in mares. Our findings suggest that at term induced foals suffer slightly greater, but not clinically significant, hypoxia, hypercapnia and acidosis than spontaneously delivered foals.

Embryo technologies in donkeys (Equus Asinus).

In industrialized countries, the donkey population had a dramatic decrease during the last century and this has brought almost all European donkey breeds to risk. Embryo technologies have already been employed as a tool for the conservation of endangered equid species (e.g. Equus Przewalskii). Today it is possible to obtain pregnancies after the transfer of donkey embryos in synchronized recipients, even though embryo transfer is not widely used as a reproductive technique in this species. So far, very few foals are born after transfer of cryopreserved embryos. To date, no pregnancies have been reported from in vitro fertilization studies. The use of the donkey as a dairy animal, as well as the increased importance of the conservation of endangered donkey breeds, could be the engine for the development and spread of embryo technologies in this species. However, funding from national and international research agencies in this field is still scarce, especially in Europe.

Pharmacokinetics of levosulpiride after single-dose administration in goats (Capra hircus) by different routes of administration.

Levosulpiride (LSP) is the l-enantiomer of sulpiride, and LSP recently replacing sulpiride in several EU countries. Several studies about LSP in humans are present in the literature, but neither pharmacodynamic nor pharmacokinetic data of LSP is present for veterinary species. The aim of this study was to assess the pharmacokinetic profile of LSP after intravenous (IV), intramuscular (IM), and oral (PO) administration in goats. Animals (n = 6) were treated with 50 mg LSP by IV, IM, and PO routes according to a randomized cross-over design (3 × 3 Latin-square). Blood samples were collected prior and up to 24 hr after LSP administration and quantified using a validated HPLC method with fluorescence detection. IV and IM administration gave similar concentration versus time curve profiles. The IM mean bioavailability was 66.97%. After PO administration, the drug plasma concentrations were detectable only in the time range 1.5-4 hr, and the bioavailability (4.73%) was low. When the AUC was related to the administered dose in mg/kg, there was a good correlation in the IV and IM groups, but very low correlation for the PO route. In conclusion, the IM and IV administrations result in very similar plasma concentrations. Oral dosing of LSP in goats is probably not viable as its oral bioavailability was very low.

Determination of Salivary Cortisol in Donkey Stallions.

Salivary cortisol provides information about free plasma cortisol concentration, and salivary sampling is a noninvasive well-tolerated procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in four donkey stallions on 13 nonconsecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected using a swab inserted at the angle of the lips, placed onto the tongue for 1 minute and returned into a polypropylene tube. Tubes were centrifuged, and at least 1 mL of saliva was aspirated from each sample and frozen at -20°C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7%, and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone, and <0.1% with testosterone, progesterone, and estradiol. The intra-assay coefficient of variation was 10.7%, the interassay variation was 8.0%, and the minimal detectable concentration was 0.01 ng/mL. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva as already reported in other species.

Post-thaw Addition of Caffeine and/or Pentoxifylline Affect Differently Motility of Horse and Donkey-Cryopreserved Spermatozoa.

To increase sperm motility, several molecules have been tested in mammals. Methylxanthines have shown effects on sperm motility, capacitation, and on in vitro fertilization processes. The aim of the study was to evaluate if the post-thaw addition of caffeine and/or pentoxifylline changes motility parameters of cryopreserved stallion and donkey spermatozoa. Straws derived from 14 horses and 7 donkeys were thawed and diluted in a milk-based extender to obtain the following final concentrations: CTR (control, no additives), CAF 5 (5 mM caffeine), CAF 10 (10 mM caffeine), PTX 5 (5 mM pentoxifylline), PTX 10 (10 mM pentoxifylline), CAF-PTX (5 mM caffeine and 5 mM pentoxifylline). Samples were evaluated immediately post-thaw and after 60 and 120 minutes of incubation at 37°C. In horses, overall total motility was significantly lower in CTR than in to CAF5, CAF-PTX, PTX5, PTX10, whereas progressive motility increased only in CAF5 and PTX5 (P < .05). No differences between control and treatments were seen for donkey semen. In CTR, during the first hour of incubation horses' sperm cells showed a larger decrease than donkeys' ones in all parameters (P < .05), except for lateral sperm head displacement. Thus, post-thaw motility and velocity decreased more sharply in horses than in donkeys. Caffeine and pentoxifylline-added post-thaw were able to increase the proportion of motile spermatozoa only for stallions and not for donkeys. Whether the improvement in post-thaw motility of equine spermatozoa may have an effect on in vivo or in vitro pregnancy rates remains to be determined.

Effect of the administration of alfaprostol 3 or 6 days after ovulation in jennies: ultrasonographic characteristic of corpora lutea and serum progesterone concentration.

Donkey jenny's corpus luteum (CL) response to PGF2α analogues has not been investigated in depth. Aim of this study was to evaluate the donkey jenny's corpus luteum (CL) ultrasonographic characteristics (diameter, area and vascularized area) by B-Mode, Color Doppler and serum progesterone concentration ([P4]) after treatment with the prostaglandins F2α analogue alfaprostol at day 3 or day 6 after ovulation (groups PG3 and PG6, respectively). [P4] was positively correlated (P < 0.0001) with CL diameter: r2 = 0.17; area: r2 = 0.21 and vascularized area: r2 = 0.54. The interovulatory interval was significantly reduced in the PG6 group (15 ±â€¯1.8 days), compared to the control group (24.5 ±â€¯2.9 days; P < 0,05), while there were no significant differences in interovulatory interval between PG3 (21.7 ±â€¯7.9 days) and control or PG6 group. [P4], in the 6 jennies of the PG6 group, dropped under 1 ng/mL within 2 days after treatment, remaining under this concentration until [P4] raised again to levels comparable with those of the control group until spontaneous luteolysis. After alfaprostol administration, one of the 2 remaining PG3 group jennies showed a complete luteolysis, and the other one underwent a partial luteolysis and ovulated in diestrus.

Variables affecting semen quality and its relation to fertility in the dog: A retrospective study.

Although semen analysis is routinely used to evaluate the male reproductive potential in the canine species, only a few authors have attempted to relate semen characteristics to donor traits, such as age or weight, and fertility. This study evaluated the effects of dogs' age (young: 0-24 months, adult: 25-84 months, old: >84 months), size (small: <15 kg, medium: 16-40 kg, large: >40 kg) and reason for semen collection (evaluation, artificial insemination, cryopreservation or research purposes) on the seminal characteristics and related the main seminal characteristics with fresh AI results. Overall, 251 semen examinations were performed from 140 dogs presented between 2000 and 2015 at the University of Pisa Veterinary Teaching Hospital. Conventional semen parameters (semen volume, sperm concentration and total number, subjective motility and sperm morphology) were evaluated. Overall, 227 (90.4%) samples contained spermatozoa. Azoospermic samples resulted from complete (n = 13, 9 dogs), or incomplete ejaculations (n = 11, 8 dogs), based on seminal alkaline phosphatase concentration and/or ancillary examinations. Reason for semen collection had a significant effect on semen volume, sperm total number and sperm concentration. The proportion of normal spermatozoa was lower in dogs presented for semen evaluation (P < 0.05). Dog size affected ejaculate volume and total sperm number, which was lower in small dogs compared to medium (P < 0.01 and P < 0.05, respectively) and large-sized dogs (P < 0.01). Age had no influence on volume, sperm number or motility. The proportion of normal spermatozoa was higher in young animals than in old ones (P < 0.05). There was a significantly higher proportion of midpiece defects in old dogs compared to young ones (P < 0.01). Total sperm number, motility and proportion of morphologically normal spermatozoa in the semen used in AIs resulting in a pregnancy were 627.6 × 106, 83.9% and 64.9%, respectively, all significantly higher than in the unsuccessful AIs, where they were 389.4 × 106, 66.5% and 42% (P < 0.05). In conclusion, age, size and reason for semen collection may affect main sperm parameters and must be taken into consideration during the interpretation of a routine semen evaluation. In turn, altered semen parameters may affect fertility after artificial insemination.