20 Development and Oct4/Cdx2 gene expression of Puma concolor, Leopardus geoffroyi, and Panthera onca hybrid embryos produced using domestic cat oocytes

Even though knowledge in sperm cryopreservation of endangered felids advanced in recent years, very little is known about suitable protocols to cryopreserve sperm from Leopardus geoffroyi (LG). In the present study, sperm obtained by electroejaculation from 5 different males were cryopreserved in either a Tes-Tris- or a lactose-based diluent (Gañan et al. 2009 Theriogenology 72, 341-352) with modifications in the freezing process using a one-step method: straws were placed horizontally on a metal rack, 4cm above the surface of liquid nitrogen in a styrofoam box, and kept for 10min before plunging them in LN. The objectives were to (1) compare in vitro motility and acrosome status of LG sperm cryopreserved in both extenders and (2) test functionality of LG sperm cryopreserved in both extenders through their ability to fertilize mature domestic cat oocytes. Straws were thawed by exposing them to air for 10s and then immersing them in a water bath at 37°C for 30s. The contents of the straws were poured into a sterile 1.5-mL microtube prewarmed to 37°C. The sperm suspension was diluted (1:3 vol/vol) by the slow (drop by drop) addition of a modified Tyrode’s solution. Sperm parameters, percentage of motile spermatozoa, and quality of motility were assessed and sperm motility index (SMI) was calculated as follows: [% motile sperm+(quality×20)]/2. Acrosome integrity was assessed by staining with Coomassie brilliant blue. For IVF, in vitro-matured domestic cat oocytes (n=238 Tes-Tris, n=239 lactose) were co-incubated with 0.5×105 motile spermatozoa/mL under 5% CO2 in air at 38.5°C for 18-20h (Pope et al. 2006 Methods Mol. Biol. 254, 227-244). Presumptive zygotes were cultured in vitro in 50-µL drops of modified Tyrode’s medium at 38.5°C in 5% CO2, 5% O2, 90% N2 atmosphere. Cleavage was assessed 48h postfertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Data was analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), significant at P<0.05. Results, mean (±standard error of the means), showed that SMI and acrosome integrity (pre- and post-thawing) were similar for both extenders: prethawed (SMI=56±3.3v. 59±5.5; acrosome integrity=88±3.0% v. 90±2.0%), and post-thawed (SMI=46±5.0v. 44±7.0; acrosome integrity=57±7.5% v. 68±2.4%) Tes-Tris v. lactose, respectively. For IVF, results showed a high cleavage rate in both groups (117/238, 49% v. 117/239, 49%), and a high development to morula (96/238, 40% v. 94/239, 39%) and to the blastocyst stage (61/238, 26% v. 51/239, 21%) for all males Tes-Tris v. lactose, respectively. There were no significant differences between groups at any development stage. In conclusion, we found that both extenders can be used to cryopreserve LG sperm maintaining functional conditions and that fertilizing capacity can be tested using in vitro-matured domestic cat oocytes.

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

In vitro embryo production (IVP) in the domestic bitch is important for conservation of endangered canids. Compared with various domestic animals, the development of assisted reproductive technologies (ART) in the dog has lagged behind, mainly due to the low percentage of oocytes that can reach metaphase II (MII) stage after in vitro maturation (IVM). Beneficial effects of l-carnitine (LC) on embryonic development in culture have been reported in many mammalian species; however, no studies have been conducted in dogs. The aim of the present study was to investigate the effect of LC supplementation during IVM of canine oocytes on nuclear maturation, fertilization status, and pre-implantation development following IVM/IVF. Cumulus-oocyte complexes (COC) were collected by slicing ovaries obtained from dogs (n = 20, 1 to 6 years of age) after ovariohysterectomy. The COC were subjected to IVM for 72 h in a medium (TCM-199) supplemented with LC at different concentrations (0.1, 0.3, 0.6, 1.0, or 2.0 mg mL−1) or without LC supplements (0 mg mL−1; control). Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and presumptive zygotes were cultured in SOF medium for 7 days. Frequencies of nuclear maturation (72 h post-IVM), fertilization rates (18 h post-insemination), and embryo development (Days 2 to 5 post-insemination) were evaluated. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. Supplementation of IVM medium with 0.3 or 0.6 mg mL−1 LC significantly improved (P ≤ 0.05) maturation (35.4% and 41.4%) and fertilization (21.3% and 25.8%) rates compared with the controls and with other LC-supplemented groups; values ranged from 20.1% to 25.0% for maturation and from 12.1% to 14.6% for fertilization. Cleavage (2- to 16-cell stages) was significantly higher (P ≤ 0.05) in the 0.6 mg mL−1 LC supplemented group than the 0.3 mg mL−1 supplemented group (16.3% v. 13.3%). These values were significantly higher (P ≤ 0.05) than those in other groups. Interestingly, 4.5% of IVM/IVF oocytes were developed to morula in 0.6 mg mL−1 LC supplemented group which was significantly higher (P ≤ 0.05) than those developed in the 0.3 mg mL−1 supplemented group (1.0%). No embryos developed beyond the 2- to 16-cell stage in the rest of the groups. In conclusion, l-carnitine supplementation during IVM is particularly efficient in improving nuclear maturation and pre-implantation embryo development of canine oocytes after IVF. These outcomes are important for the improvement of IVM conditions that can advance the efficiency of ART in dogs.

The effect of seminal plasma on alpaca sperm function

Influence of living status (single vs. paired) and centrifugation with colloids on the sperm morphology and functionality in the clouded leopard (Neofelis nebulosa)

An improved understanding of reproductive physiology in nondomestic bovids is necessary for the development of assisted reproductive technologies (ARTs) for use in the conservation of endangered bovids. In this study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls in South Africa. Our objectives were 1) to characterize the quality of epididymal spermatozoa, 2) evaluate the effectiveness of a cryopreservation protocol, and 3) compare postthaw sperm longevity (motility, viability, and acrosomal integrity) and functionality in two culture media with two capacitation reagents (caffeine and heparin). Following recovery, spermatozoa were diluted in EQ extender, slow-cooled, and frozen in the presence of 5% glycerol. Thawed spermatozoa were separated on a Percoll gradient and diluted in fertilization media (SOF for fertilization [SOFfert]; 0.6% BSA, 0.0 mM glucose, 25.0 mM NaHCO(3)) or modified SOFfert (1.2% BSA, 1.5 mM glucose, 37.0 mM NaHCO(3)) and either heparin or caffeine, and incubated for 6 h. Spermatozoa from these species maintained an average of 64% initial motility after thawing. Incubation medium and capacitation reagent had species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa, suggesting ART procedures need to be optimized for each species. Springbok spermatozoa were also shown to be competent for in vitro fertilization. Information from this study concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest will be useful in the development of ART for the conservation of these and other species of bovids.

There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P < .004). Cryopreservation in either Botu-Crio or INRA 96 resulted in a decline (P < .05) in percent sperm motility and acrosomal integrity. Sperm forward progression remained unaffected immediately after thawing in INRA 96 but continued to decline over time. These results characterize, for the first time, the ejaculate traits of the tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25 degrees C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

Development of assisted reproductive technologies for the critically endangered red wolf (Canis rufus) is crucial to the maintenance of genetic diversity to support species recovery. Towards this goal, a cryopreservation protocol has previously been developed for red wolf sperm; however, the ability of the gametes to undergo capacitation has not been assessed in this species. Previously, we have shown that oviductal extracellular vesicles (oEVs) improve cat sperm motility and fertilizing ability. The objectives were to (1) compare the effects of culture media on motility and acrosomal integrity of fresh sperm, and select the best medium that can be used in a capacitation protocol; (2) identify potential biomarkers for sperm cryo-tolerance; and (3) determine the influence of canine oEVs on sperm survival and motility post-thaw. In Study 1, sperm were collected by electro-ejaculation from adult red wolves (n=8) and immediately cryopreserved in TRIS-egg yolk buffer with 8% glycerol or incubated for 18h in 5% CO2 and 38.5°C in one of the following media: canine capacitation medium (CCM), FERT-TALP (FERT), NCSU, synthetic oviductal fluid (SOF) and TRIS. At 0, 1, 2, 3, 4, and 18h, sperm were evaluated for total motility and acrosome integrity (FITC-PNA). In Study 2, sperm with high (&amp;gt;80%, HM, n=2 wolves) and low (&amp;lt;15%, LM, n=2 wolves) motility post-incubation at 4°C in the cryopreservation medium for 18h were subjected to proteomic analysis. In Study 3, oviducts were collected from domestic dogs (1-9 years, n=12) after elective spaying, and oEVs from various stages of the oestrous cycle [early follicular (EF), late follicular (LF), early luteal (EL), and late luteal (LL)] were isolated using the Total Exosome Isolation kit (Invitrogen). Frozen-thawed red wolf sperm (n=4 males) were incubated with 30×106 oEVs in non-capacitating CCM, and assessed as in study 1 at 0, 0.5, 1, 2, 3, 4, 6, 8, and 10h. Data were analysed using a paired samples t-test with 95% CI (Prism8, GraphPad Inc.). Sperm incubated in CCM and NCSU had higher motility than those in FERT, SOF, and TRIS after 2h of incubation and onward (2 h: 65±6, 68±6, 42±10, 57±8, and 43±5; 3 h: 60±9, 63±8, 36±11, 46±9, and 34±6; 4 h: 60±9, 60±10, 30±10, 43±8, and 20±5; 18 h: 12±7, 15±7, 9±5, 3±2, and 0, respectively; P&amp;lt;0.05). After 1h of incubation, samples incubated in CCM, NCSU, and SOF had a higher number of sperm with intact acrosomal membranes (P&amp;gt;0.05) than other treatments. A total of 179 proteins were identified, of which 129, including those regulating energy metabolism and mitochondrial mediated apoptosis, were differentially expressed between HM and LM. Preliminary data from Study 3 suggested that thawing and incubating sperm in the presence of LF, EL, and LL oEVs improved sperm motility. In conclusion, CCM and NCSU sustained sperm survival after invitro incubation and could be candidates for invitro fertilization studies in the red wolf. Data generated from sperm proteomic analysis provided insights into cellular pathways regulating sperm cryo-sensitivity. Finally, we demonstrated the potential of oEVs in improving wolf sperm survival post-thawing.

The Puma concolor population has been decreasing during the last 30 years. Semen cryopreservation of this species has been accomplished successfully and offers the possibility of preserving endangered species. We previously showed that fertilizing capability of wild felid spermatozoa can be evaluated using intracytoplasmic sperm injection (ICSI) with in vitro-matured domestic cat oocytes (Moro et al. 2014 Reprod. Domest. Anim. 49, 693-700). Due to the lack of homologous oocytes, we evaluated the capability of the Puma concolor sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. In the present study, cryopreserved sperm obtained by electroejaculation from five different males were used for IVF of in vitro-matured (IVM) domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing in a 37°C water bath for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube pre-warmed to 37°C. The sperm suspension was diluted (1:3 v/v) by the slow (drop-by-drop) addition of a modified Tyrode’s solution. For IVF, IVM oocytes (n = 370) were co-incubated with 0.5 × 105 motile spermatozoa mL−1 in an atmosphere of 21% O2 in air at 38.5°C for 18 to 20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C. Cleavage was determined at 48 h post-fertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Results (mean ± SEM) showed a high cleavage rate (179/370, 49.0 ± 4.0%), and a high development to morula stage (137/370, 34.4 ± 7.2%), and to blastocyst stage (94/370, 23.4 ± 4.7%) for all males. These results indicated that Puma concolor spermatozoa can induce domestic cat oocyte activation and development to blastocyst stage in similar rates to domestic cat homologous IVF: IVM oocytes (n = 291), cleavage rate (199/291, 67.1 ± 6.1%), development to morula stage (144/291, 47.8 ± 4.9%), and to blastocyst stage (86/291, 30.1 ± 1.6%). In conclusion, we demonstrated that domestic cat oocyte can be used to evaluated cryopreserve sperm samples from another felid species.

Once nearly extinct in the wild, the southern white rhinoceros is currently listed as near threatened by IUCN. This status is likely to change as poaching continues to escalate. To preserve the species’ current genetic diversity, cryopreserving and biobanking white rhinoceros sperm is imperative. The horse is the closest domestic relative of the rhinoceros and a useful model for the development of assisted reproductive technologies, including semen cryopreservation. Two equine semen cryopreservation protocols were compared to a common rhinoceros freezing method. Semen was collected from a single male on 3 occasions by electroejaculation. Initial semen parameters were 86% motility; speed 3.2 (scale 1-5); 89% plasma membrane integrity; and 95% intact acrosomes. Semen was extended 1:1 in INRA 96 (IMV Technologies, L’Aigle, France) before centrifugation at 400×g for 10min. Supernatant was removed and the sperm pellet was subjected to 1 of 2 treatments: resuspension in 500µL of either BotuCrio (Botupharma, Botucatu, Brazil) or Cryomax (ARS Inc., Chino, CA, USA), both containing a proprietary combination of glycerol and an amide as cryoprotectants. Following a 40-min cool at 4°C, extended semen was frozen in vials at a cooling rate of 30°C/min for 3min before LN submersion. Control semen was extended 1:1 in TEST-Y buffer without cryoprotectant and cooled for 2.5h before adding glycerol to a final concentration of 4%. Extended sperm (500µL) was frozen in vials at 12.5°C/min for 15min before LN submersion. Initial motility score (IMS;% motile×speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded after extension. All vials were thawed at 37°C for 60s and the cryoprotectant was removed by centrifugation. Sperm pellets were resupended in M199+HEPES and sperm was evaluated for the characteristics described above at 37°C at 0, 30, and 60min (T0, T30, T60) post-thaw. All data are expressed as a percentage of initial (%IMS,%IPL, and%IAC) to account for the differences in sperm parameters between ejaculates. Cryopreservation protocol significantly affected%IMS at T0 (P=0.0131, Table 1). Although the differences were significant only at T0, sperm frozen in Botucrio or Cryomax tended to maintain a higher%IMS than the control freeze at all time points. However, sperm frozen in Cryomax lost a greater percentage of%IMS over time (67% from T0 to T60v. 44 and 46% for Botucrio and TEST-Y, respectively). Cryopreservation protocol did not affect%IAC or%IPL at any time point, but again Cryomax and Botucrio tended to be higher than TEST-Y. This study indicates that rhinoceros sperm may suffer less cryodamage in Botucrio or Cryomax frozen at 30°C/min than in the conventional TEST-Y frozen at 12.5°C/min. Table 1.Percent of initial motility score (IMS), plasma membrane integrity (IPL), and acrosome integrity (IAC) at 0, 30, and 60min post-thaw (T0, T30, and T60, respectively)

Context Porcine gametes require energy for the physiological processes that allow fertilisation. Succinate dehydrogenase (SDH) plays a pivotal role in both, the tricarboxylic acid (TCA) cycle and the respiratory chain. Aims The aim of this work was to study the participation of SDH in the in vitro oocyte maturation, sperm capacitation and acrosome reaction in porcine species. Methods Cumulus–oocyte complexes (COCs) from abattoir-derived porcine ovaries were collected by aspiration and were incubated in maturation media, with the addition of increasing concentrations (0, 1, 5 and 10 mM) of malonate (a specific inhibitor of SDH). Nuclear maturation and cytoplasmatic maturation were analysed. Semen samples were incubated for 2 h in capacitating medium with 40 mM sodium bicarbonate, as sperm capacitation inducer, and the addition of increasing concentrations of malonate (0, 1, 5 and 10 mM). Sperm capacitation state and true acrosomal reaction were evaluated. SDH activity was determined in sperm and oocyte extracts by the spectrophotometric method. Key results The addition of 10 mM of malonate decreased both nuclear and cytoplasmic maturation rates (P &amp;lt; 0.05) without affecting COC viability (assessed using fluorescein diacetate). A lower level of capacitation (induced by bicarbonate) and acrosome reaction (induced by follicular fluid) was observed with the addition of 5 mM of malonate (P &amp;lt; 0.05) without affecting motility and viability of sperm at this concentration. The activity of SDH was 0.35 ± 0.1 × 10−5 and 2.37 ± 0.9 × 10−5 U/COC for immature and in vitro matured COC extracts (P &amp;lt; 0.05) respectively, and 0.44 ± 0.16 U/1010 sperm for boar sperm extracts. Conclusions In conclusion, because it has been proposed that aerobic and anaerobic metabolic pathways of cells are changed depending on the oxygen availability and the composition of metabolic substrates in their environment, our results suggest that energy obtained through the mitochondrial respiration (TCA cycle and oxidative phosphorylation) is necessary to support oocyte maturation, sperm capacitation and acrosome reaction in the porcine species. Implications The study of enzymatic activity in gametes is essential for understanding the mechanisms that control the energy production required to achieve successful fertilisation. This knowledge has significant implications for the development of assisted reproductive technologies.

To compare the in vitro fertilization (IVF) and production (IVP) of embryos from low and high antral follicle count (AFC) heifers, AFC were determined on 106 heifers using transrectal ultrasonography. Ten heifers with the lowest AFC (avg. 13.2) and 10 heifers with the highest AFC (avg. 27.4) with evidence of oestrous cyclicity were synchronised with 2 injections of prostaglandin F2α (PGF2α); half were harvested on Days 5 to 6 and half on Days 15 to 16 of the oestrous cycle. The IVF procedures included protocols for semi-defined media and were as described (IVP Protocol, P. J. Hansen’s Laboratory, University of Florida, Gainesville, FL, USA). Cumulus-oocyte complexes (COC) from follicles less than 8 mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24 h. Matured COC were fertilised using thawed frozen semen from a crossbred bull that was purified using Percoll gradient separation procedures. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1 × 106 spermatozoa per mL. About 24 h later, presumptive zygotes were placed in microdrops of development medium under oil, and cultured (5% CO2, 5% O2, balance N2, 38.5°C). On Days 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the mixed procedure of SAS (SAS Institute, Cary, NC, USA) and the model included the effects of collection day, group (high or low AFC), and their interaction. More COC (P &lt; 0.0005) were collected from high than low AFC heifers (30.3 v. 9.3 ± 3.12 per heifer). Both the number and percentage of COC that cleaved had an interaction between collection day and group (P &lt; 0.03). The interaction appeared to be due to low cleavage and development rates on 1 of 4 collection days (appeared not related to day of oestrous cycle). Although high compared to low AFC heifers had more COC that cleaved (18.7 v. 4.4 ± 1.84 per heifer), the percentage of cleaved COC did not differ (59.2 v. 49.8 ± 3.36%). There were no significant differences between high and low AFC heifers in the number of blastocysts (3.1 v. 0.6 ± 1.21 per heifer) or in the percentage of COC that developed to blastocysts (8.8 v. 5.2 ± 3.60%). In previous replicates (years), we reported that high AFC heifers had more COC collected, more COC that cleaved, and more COC that developed to blastocysts than low AFC heifers. In contrast, in this study numbers of COC that developed to blastocysts did not significantly differ between high and low AFC heifers. Additionally, the percentage of COC that cleaved, and that developed to blastocyst have been similar between high and low AFC heifers. Therefore, high compared to low AFC heifers may produce more IVP embryos; however, AFC does not appear to affect the competence of an oocyte to develop and mature to the blastocyst stage.

Simple SummaryThe kodkod (Leopardus guigna) is a small wild felid native to Chile, whose population is currently in decline. Assisted reproductive technologies (ARTs) offer valuable tools for the genetic preservation of endangered species, including the kodkod. Conservation strategies for the kodkod have included cryobanking and the generation of cloned embryos through interspecific somatic cell nuclear transfer (iSCNT). However, the reproductive physiology and embryo development of this species are poorly understood. Here, we applied optimized protocols developed for domestic cat oocytes to evaluate the in vitro oocyte maturation (IVM) and in vitro culture (IVC) of embryos from kodkod. Immature cumulus–oocyte complexes (iCOCs) were collected from a female kodkod, and their morphology and developmental competence were evaluated. Immature kodkod oocytes were smaller than domestic cat oocytes. Kodkod oocytes were able to mature in vitro and develop to the blastocyst stage following chemical activation. Our work demonstrates that the protocols used to manage domestic cat oocytes can be successfully adapted for IVM and in vitro embryo production (IVP) in the kodkod. Our research provides a foundation for improving ARTs aimed at conserving this endangered felid and enhancing our understanding of its reproductive biology.The kodkod (Leopardus guigna) is a vulnerable wild felid native to South America whose population is steadily declining. ARTs offer valuable tools for the preservation of its genetic diversity. Our study provides the first evaluation of the morphological and functional acquisition of competence in kodkod oocytes using protocols previously established for domestic cat oocytes. In total, 29 iCOCs were obtained from the ovaries of a single juvenile female kodkod that deceased in a wildlife rehabilitation center. Based on morphological criteria, 13 oocytes were selected for IVM and subsequently evaluated for developmental competence following parthenogenetic activation (PA) and in vitro culture (IVC). Kodkod oocytes appear to be smaller and have a thinner zona pellucida compared to those of domestic cat oocytes. These kodkod oocytes demonstrated the ability to mature in vitro, underwent cleavage, and developed in vitro to the blastocyst stage by day 9. Here, we show that protocols to manage domestic cat oocytes and embryos can support kodkod in vitro oocyte maturation, activation, and in vitro embryo development. However, given that the results were obtained from a single individual and the protocols were tested in a limited number of oocytes, further studies involving additional specimens are essential to validate these observations and refine ART applications for kodkod conservation.

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500µL of semi-defined IVM medium for 22h at 38.8°C in a humidified atmosphere with 5% CO2. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5M sucrose for 30s. Afterwards, COCs were loaded in &amp;lt;1µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3mL of 1M sucrose in BM for 2min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500µL of 0.5M sucrose in BM for 2 min; (2) 500µL of 0.25M sucrose for 2 min; (3) 500µL of BM for 2min. Then, COCs from each group were transferred to 250µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 106 sperm mL−1 for 18h. At Day 1, all presumptive zygotes were cultured in 25µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO2 and 5% O2. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P&amp;gt;0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P&amp;lt;0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P&amp;gt;0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device. This research was funded by CAPES/COFECUB (#88881.142966/2017-01).