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.

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.

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.

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%.

Gross and histological findings in the canine placenta and amnion at term: What's normal, abnormal or pathological?

The canine placenta is an underexamined organ. Placental abnormalities can affect foetus development and may be responsible for a low weight of the infant at birth; however, knowledge on their clinical significance in the canine species is limited. We aimed to describe macroscopic and microscopic findings in the canine placenta and amnion at term in clinically uncomplicated pregnancies and to evaluate their relationship with birth weight of healthy puppies. During natural delivery or C-section, the birth weight of 82 puppies was recorded, 72 placentas and 66 amnions were recovered. The foetal and maternal surfaces of the placental girdle, marginal haematoma and amnion were evaluated. Each gross finding was recorded, morphometrically assessed and sampled for histological diagnosis. Furthermore, specimens of placenta and amnion were collected from representative areas and microscopic deviations from normal structure were evaluated in haematoxylin and eosin sections. Gross examination revealed 'abnormalities' in the 75.4% of the collected placentas. Necrosis was the gross change most commonly observed in the placental girdle (72.5%). Congestion (17.4%) and clotted blood/fibrinoid material (2.9%) were also observed. No gross changes of either the marginal haematoma or the amnion were recorded. Histologically, placental girdle showed necrosis (62.3%), mineralization (52.2%), congestion (36.2%) and neutrophilic infiltration (27.5%). Marginal haematoma exhibited mineralization (11.6%) and neutrophils (29%), while necrotic foci were rarely observed (4.3%). In the amnion, the most frequent alteration observed was hypertrophy of the epithelium (35.9%) followed by oedema (31.2%), mineralized foci (28.1%), fibrosis (23.4%), congestion (15.6%) and more rarely neutrophils (12.5%). Puppies' birth weight was not statistically affected by either gross or histological abnormalities. Our study revealed that macroscopic and microscopic 'abnormalities' of the placenta and amnion may be common in uncomplicated pregnancies at term; however, no implications on puppies' birth weight were observed. Deviations from 'normal' morphology of canine foetal adnexa warrant further investigation to assess their clinical implications if present.

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.

Report on a Milking Mule: Milk Qualitative Characteristics during Lactation.

Despite their inability to reproduce naturally, mules can host embryos and be surrogate dams. The aim of this investigation was to increase our knowledge of the qualitative composition of mule's milk and its variations throughout the whole lactation period-namely, from 6 h after foaling to 180 days in milk (DIM). Milk was obtained from a mule dam that had foaled after receiving a mule embryo transfer. For each sample, the gross, mineral, and fatty acid composition was evaluated. The average quality of the mule milk was as follows: protein 1.97 g 100 mL-1, fat 0.90 g 100 mL-1, and ash 0.39 g 100 mL-1. Saturated fatty acids made up, on average, 50.00 g 100 g-1 of fat. Monounsaturated and polyunsaturated fatty acids made up half of the total fatty acid content (31.80 g 100 g-1 and 18.2 g 100 g-1 of fat, respectively). Linoleic acid and linolenic acid were the main polyunsaturated fatty acids in the milk. The milk composition changed throughout lactation. Dry matter, protein, fat, and ash decreased significantly from early lactation (6 h to 14 DIM). The n3 polyunsaturated fatty acids decreased at the end of lactation. The changes in milk composition throughout lactation are probably due to adaptations to the growth requirements of the foal.

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.

Apgar Score, Clinical, Hemato-Biochemical, and Venous Blood Gas Parameters in a Cohort of Newborn Mule Foals: Preliminary Data.

The study aimed to assess Apgar scores and collect data on clinical, hemato-biochemical, and venous blood gas parameters in seven mules during their first 48 hours of life. Apgar scores, heart rate (HR), respiratory rate (RR), body temperature (BT), body weight (BW), time to achieve sternal recumbency, stand, nurse, and pass meconium were recorded. Blood samples were collected at birth (T0), 6 (T6), 12 (T12), 24 (T24), 48 (T48) hours of life. Data distribution was verified and differences in laboratory parameters over time assessed. One foal had an Apgar score of 6/8, thus excluded from the statistical analysis. Three foals had an Apgar score of 7/8, three had a score of 8/8. All foals showed suckling reflex within 20 minutes and raised the head within 1 minute; six foals reached sternal recumbency within 2 minutes. Times to stand and nurse were 38.7 ± 13.4 and 72.3 ± 30.5 minutes, respectively. Five foals passed meconium spontaneously in 416.3 ± 401.8 minutes. The mean HR, RR, and BT values were 92 ± 16.1 bpm, 50.2 ± 5.9 bpm, and 37.6 ± 0.3°C, respectively. Creatinine and lactate decreased from birth to 48 hours. Blood glucose increased from 12 hours. Mg increased from 12 to 24 hours. Compared to horses and donkeys, mules showed an intermediate BW, shorter time to stand and nurse, longer time to pass meconium, intermediate HR and lower RR. Compared to donkey mules showed lower BT. Other dissimilarities from the parental species were found in laboratory parameters. Our findings emphasize the need for reference ranges for a comprehensive evaluation of newborn mules.

Embryonic resorption rates at canine pregnancy diagnoses: A retrospective evaluation.

Pregnancy diagnosis in canines is generally performed during embryonic phase, between 19 and 35 days of gestation. At this stage embryonic resorptions can be observed, which, according to the literature, affects 11-26% of conceptuses and 5-43% of pregnancies. Resorption has been hypothesized as a physiological event in uterine overcrowding, however other factors may be involved, such as infectious or non-infectious diseases. This study aimed to retrospectively evaluate the incidence of embryo resorption at ultrasonographic pregnancy diagnosis in different dog breeds, and to identify the main factors determining the occurrence of the resorption sites. 95 pregnancy diagnoses were performed 21-30 days post-ovulation by ultrasound examination on 74 different animals. Breed, weight, and age of the bitches were recorded, and the reproductive anamnesis was collected from their medical records. The overall pregnancy rate was 91.6%. In 48.3% of pregnancies (42/87), at least one resorption site was visible, and embryonic resorption rate was 14.2% (61 resorption sites/431 total structures). Binary logistic regression showed a significant effect of age (P < 0.001), but not the size of the litter (P = 0.357), nor the size of the mother (P = 0.281) or any previous reproductive problems (P = 0.077). Age was significantly higher in pregnancies with resorptions than in normal ones (60.88 ± 18.24 and 40.27 ± 15.74 months, respectively, P < 0.001). The embryonic resorption rate was in line with previous findings, while the incidence of affected pregnancies was higher. Although resorptions may occur physiologically in pregnancies with large litters, a relationship between embryo resorption and litter size was not identified in our sample group, while aging increased the resorption rates. This, together with the occurrence of repeated embryonic resorptions in some bitches included in the study, suggests how resorptions could also be the result of pathological events. The underlying mechanisms and other factors that may be involved need further clarification.

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.

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.

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.

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.

Age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs.

There are age-related changes in testicular anatomy and physiology whereby there are modifications of sperm production and reproductive hormone functions. Effects of age on testicular microanatomy are well documented in humans, while there is limited understanding of these changes in dogs. The aim of this study was to evaluate age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs. Dogs (n = 32) were divided into four age groups: peripubertal (n = eight), relatively younger (n = seven), reproductively mature (n = seven) and relatively older (n = ten). Picrosirius Red stained sections were used for morphometrical analysis of testicular tissues, while the characteristics of seminiferous epithelium were assessed using a modified Johnsen scoring system for haematoxylin and eosin stained sections. Seminiferous epithelium and seminiferous tubule area increased from peripuberty to reproductive maturity, indicating there were changes during sexual maturation and subsequently there were decreases with further aging. There was a similar age-related trend for changes in seminiferous epithelium height with values being greatest in reproductively mature dogs; while there were no age-related differences in tubular diameter. Collagen content in the testicular interstitium gradually decreased from peripuberty to the age when dogs were reproductively mature and there were subsequent increases in relatively older dogs, thus, there was an association between the extent of testicular fibrosis and senescence. There was a decrease in spermatogenetic functions from relatively younger to older ages. Further investigations are warranted to establish mechanisms responsible for age-related changes of testicular morphology and related clinical implications.

Relationship between placental characteristics and puppies' birth weight in toy and small sized dog breeds.

Puppy neonatal mortality may be related to low birth weight, which has been shown in humans to be linked to placental factors. The relation between the newborn puppy and the placental characteristics has been poorly investigated in dogs. Twenty bitches, 9 toy-sized (i.e. < 5 Kg) and 11 small-sized (i.e. 5 to 10 Kg), were included in this study. During natural delivery or c-section, puppies were identified and their order of birth, sex and weight were recorded. Puppy weights were registered at birth and daily until Day 6. Placentas were weighed after removal of extraplacental adnexa, after which a photo was taken and morphometrically assessed. The Total Placental Area (TPA) and the Transfer Zone Area (TZA) were calculated and their surface expressed in mm2. Immunohistochemistry with monoclonal mouse anti CD31 antibody was used to identify fetal and maternal vessels in the placental labyrinth zone. A vascularization index (VI) was determined for each placenta and the Total Vascular Area (TVA) was estimated. Puppies' birth weight correlated positively with placental weight (P < 0.001, r = 0.689). A positive correlation was found between the puppies' birth weight and TPA (P < 0.001, r = 0.786), TZA (P < 0.001, r = 0.772), and TVA (P < 0.001, r = 0.482). A positive correlation was also found between placental weight and TPA (P < 0.001, r = 0.661), TZA (P < 0.001, r = 0.583), and TVA (P < 0.001, r = 0.333). In the small-sized breeds, the placentas of low-weight puppies were lighter and had a smaller TZA and TVA (P < 0.05). The VI was higher in the placentas of the toy-sized compared to small-sized bitches (P < 0.01). No effect of parity, litter size, or sex of the puppy was observed on birth weight or growth rates on Day 6. The growth pattern of low-weight puppies did not differ from that of the other puppies during the first 6 days of life. As in humans, placental weight, the extension of the transfer zone and placental total vascular area correlates closely with the puppies' birth weight in normal pregnancies. Our data could represent reference values for placental weight, TZA, TVA and VI in toy and small-sized dog breeds.

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.

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.

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.