bluuebunny/crossref_metadata_2025 huggingface dataset:
143 Effect of corpus luteum on recovery rate, quality, and in vitro developmental competence of oocytes in Bos indicus dairy cows
Authors: Saad M. , Sarwar Z. , Saleem M. , Arshad U. , Shahzad M. , Mushtaq M. H. , Riaz A. , Ahmad N.
Published: December 2018

Assisted reproductive technologies have modernized the dairy industry for the rapid multiplication of superior genetic traits. However, the exploitation of genetics through in vitro embryo production in farm animals has been struggling during the last 3 decades. The presence or absence of a corpus luteum (CL) on ovaries from which cumulus-oocyte complexes were recovered has been shown to affect the quality of oocytes and in vitro embryo production outcome in Bos taurus cows. Similar information is lacking in Bos indicus cows. Therefore, the objectives of the present study were to determine the effect of presence or absence of a CL on recovery rate, quality, and in vitro developmental competence of oocytes in Bos indicus dairy cows. This study was conducted from December 2017 to April 2018. The ovaries were harvested from a local abattoir (Bos indicus; 5- to 8-year-old cows having mixed parity with clinically normal reproductive tracts). These ovaries (n=750) were divided into 2 groups [(1) CYCLIC (n=318) and (2) ACYCLIC (n=432)] based on the presence or absence of mature CL having follicles on either left or right ovaries of slaughtered cows. Blood samples from the jugular vein were collected at slaughter. Serum concentrations of progesterone of CYCLIC and ACYCLIC cows were measured using radioimmunoassay. Mean number of recovered oocytes per ovary and serum concentration of progesterone were compared using PROC TTEST model. However, quality, maturation, cleavage, 8-cell, 16-cell, and morula rates were analysed by PROC FREQ model of Chi-squared. All the data was analysed using SAS (SAS Institute Inc., Cary, NC, USA). Results revealed that the mean serum concentrations of progesterone (4.21±0.4v. 0.5±0.2ng mL−1; P<0.05) were higher in CYCLIC as compared with ACYCLIC cows, respectively. The mean number of oocytes recovered per ovary (6.5±4.5v. 4.0±4.4; P<0.05) was higher in CYCLIC than in ACYCLIC cows. The oocytes with grade I+II quality (55.3v. 47.6%; P<0.05) were higher, whereas oocytes with grade III+IV quality (44.5v. 52.4%; P<0.05) were lower, in CYCLIC as compared with ACYCLIC cows, respectively. Furthermore, cleavage rate (70.9v. 52.8%; P<0.05) was higher in CYCLIC than in ACYCLIC cows, respectively. Similarly, the 8-cell (38.5v. 20.8%; P<0.05) and 16-cell (20.0v. 10.9%; P<0.05) stage embryos were greater in CYCLIC as compared with ACYCLIC cows, respectively. In conclusion, the presence of CL has a beneficial effect on recovery rate, quality, and in vitro embryo production outcomes in Bos indicus dairy cows. It is implied that for ovum pickup, cyclic cows should be preferred.
144 Sex ratio of in vitro-produced goat embryos
Authors: Stoltzfus M. , Wayman J. , Stilz R. , Bresnahan D.
Published: December 2018

Goats are important livestock species because they produce meat, milk, and fibre and are also easily maintainable on small farms. Although goats provide many products and consumption of goat meat is increasing in the United States, the industry lags compared with many species with regard to IVF techniques to enhance goat production. It has been demonstrated in other species that male IVF embryos tend to develop faster than those of females. This may be due to increased tolerance of male embryos to inadequate conditions, particularly, glucose concentrations in culture media. However, the sex ratio of goat embryos produced utilising IVF remains unknown. The aim of this study was to determine the sex ratio of goat embryos utilising a commercially available media suite (IVF Biosciences, Falmouth, UK). Oocytes were harvested from ovaries obtained from 2 local abattoirs and matured in vitro. Frozen sperm from 1 of 2 billy goats were randomly assigned for each round of IVF. Embryos were evaluated daily from Days (D) 6 through 9 of in vitro culture. On the day an embryo reached the expanded blastocyst stage, it was removed from culture and placed into DNA extraction buffer (PicoPureTM DNA Extraction Kit, Applied Biosystems, Waltham, MA, USA) and stored at −20 for PCR analysis, typically within one month of collection. In all unknown samples, positive male (sperm) and female (uterus) controls, the amelogenin gene was amplified and products were evaluated on a 1.5% agarose gel with ethidium bromide. Embryos with 2 bands (202 and 262bp) were classified as male, and those with 1 band (262bp) were classified as female. Embryos with no bands were not included in analysis. Embryos reached the expanded blastocyst stage on D6 (n=29), D7 (n=39), and D8/9 (n=35, combined for evaluation). A chi-squared analysis comparing the percentage of male and female embryos to the expected 50% was completed for each time point (D6, D7, D8/9), as well as overall ratios (D6-9). In total, 350 oocytes were utilised in 6 rounds of IVF resulting in a mean blastocyst rate of 32% (range 17-47%). There was no significant difference in the number of embryos that were male on D6 (55%) and D7 (46%). However, on D8/9 significantly fewer embryos were male (29% male; P=0.01). Overall, there was no significant difference (P=0.14) in the sex ratio, with 41% male and 59% female embryos. Our findings are somewhat consistent with other species, in that male goat embryos produced via IVF develop more quickly in culture conditions; however, female embryos were still able to tolerate culture conditions. Delayed blastocyst development may not necessarily be an indication of a reduced quality embryo but one that is slower to develop based on its sex. This could be due to expression of X-linked genes being unbalanced during pre-implantation embryo development stages and warrants further study. One influencer of sex ratio we are currently investigating is the impact of glucose during culture, to further understand metabolism in IVF embryos.
146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos
Authors: Hicks E. , Winn E. , Whitaker B.
Published: December 2018

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P<0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P<0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P<0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.
147 Pre-incubation of bovine sperm used for IVF accelerates the developmental kinetics of the resulting embryos and possibly their sex ratio
Authors: Kotarski F. , Zimmer B. , Wrenzycki C.
Published: December 2018

The sex ratio of newborn calves and embryos produced in vivo is ~1:1. However, numerous studies on bovine in vitro-produced embryos suggest that the sex ratio may differ from 1:1 and that the rate of development may be influenced by the sex of the embryo under certain culture conditions. The duration of sperm-oocyte interaction and sperm pre-incubation also affect the sex ratio of bovine embryos produced in vitro. It is well documented that in vitro male embryos reach the more advanced stages earlier than do their female counterparts. Selection of developmentally more advanced embryos in anticipation that they have a greater developmental capacity may be one of the underlying causes of the disproportionate number of males among offspring born after transfer of in vitro-produced embryos. The aim of the present study is to test whether a pre-incubation of sperm before IVF might improve the developmental rates and also influence the sex ratio of the resulting embryos. Bovine cumulus-oocyte complexes were recovered from abattoir-derived ovaries by the slicing method. After 24h of maturation, fertilization was realised using a standard protocol. Prior to IVF, sperm cells from 2 different bulls were treated as follows: sperm within group A were pre-incubated in IVF medium for one hour. This step was omitted for sperm in group B (control). After 19h of co-culture of COC and sperm, presumptive zygotes were cultured in SOFaa for a period of 7 days. Cleavage and developmental rates were recorded at Day 3 and 7 (Day 0=IVF). Day 7 blastocysts from all groups were sexed using bovine and Y chromosome-specific primers. Data were analysed by ANOVA. As shown in Table 1, sperm pre-incubation did not affect the cleavage and developmental rates for the individual bull (P>0.05). On average, at Day 7 of development a higher number of blastocysts was determined when embryos had been produced from pre-incubated sperm (P ≤ 0.05). This held true for both bulls. The shift in favour of male embryos was detectable in all groups of embryos, with a drastic one for bull 1 after sperm pre-incubation. In conclusion, sperm pre-incubation accelerated embryo development and possibly enhanced the proportion of male embryos, which was already shifted toward males.

Table 1.Developmental rates, developmental kinetics and sex ratio of embryos after sperm pre-incubation before IVF (mean±standard deviation)
15 The effect of different bovine oocyte recovery methods on oocyte ultrastructure pre- and post-in vitro maturation
Authors: Foster B. A. , Gutierrez E. J. , Bondioli K. R.
Published: December 2018

In current in vitro embryo production practices, method of oocyte recovery tends to be based primarily on the personal preference of the operators and availability of resources rather than on any practical understanding of the best practices for retrieving oocytes of the greatest competence for embryo production. There is little consensus on the effects of either postmortem follicular atresia or exogenous hormone-stimulated follicle development on the quality of oocytes, but it is expected that both interventions have effects. Cumulus-oocyte complexes were collected from beef cattle using 3 methods: unstimulated ovum pickup (OPU), OPU following superstimulation (FSH), and oocyte aspiration from postmortem-recovered ovaries (PM). Each collection method was repeated 3 times, with 25 individuals used in each. There were no interventions in place before oocyte recovery in either OPU or PM. In FSH, cows underwent dominant follicle removal and were treated with 200mg of FSH in 6 doses before OPU. The majority of oocytes (66 OPU, 80 FSH, and 160 PM) were fertilized and cultured, with a portion being reserved for transmission electron microscopy (2 per treatment at each of germinal vesicle and metaphase II) and a portion being reserved for a complementary study. However, oocytes collected by OPU following FSH had notable ultrastructure differences from those collected by other methods, suggesting a potential effect of follicle stimulation on both the development and cytoplasmic maturation of oocytes. Although oocytes from all recovery methods and at both maturation levels exhibited mitochondria primarily in the hooded conformation, both OPU and FSH oocytes, at germinal vesicle and metaphase II, had several anomalous mitochondria exhibiting a lobulated, budding-type shape. This is potentially indicative of these oocytes having failed to complete the mitochondrial division phase before recovery. An average of 11.6, 10.6, and 11.9 oocytes per individual were recovered from PM, FSH, and OPU, respectively, resulting in oocyte recovery of 55, 61, and 57% of aspirated follicles, respectively (P>0.05). Fewer oocytes from OPU developed to morulas compared with other recovery methods (chi-square; P<0.01; 24% PM, 21% FSH, and 3% OPU). Cleaved embryos from OPU had decreased blastocyst production rates at 14% compared with 59% (PM) and 55% (FSH; chi-square; P<0.01). Overall results suggest that although superstimulation may affect oocyte ultrastructure, this does not have a negative effect on oocyte competence, though further investigations are required. Oocyte recovery rates were not affected by recovery method, but oocytes recovered by unstimulated OPU had decreased developmental competence compared with those recovered either postmortem or following superstimulation.
151 Effect of season on the in vitro fertilizing ability of frozen - thawed bovine spermatozoa
Authors: Sabes-Alsina M. , Wallgren M. , Sjunesson Y. , Lundeheim N. , López-Béjar M. , Morrell J. M.
Published: December 2018

Previous research indicated that the season during which oocytes are harvested affects quality of in vitro-produced embryos (Gupta et al. 2016 Anim. Reprod. Sci. 164, 162). In our own studies, sperm kinematics, membrane integrity, acrosome status, mitochondrial potential and reactive oxygen species were affected by season of semen collection (Sabés-Alsina et al. 2017 Vet. Rec. 180, 251). The aim of this study was to investigate effect of season of semen collection on in vitro fertilizing ability and embryo development of the same sperm samples from the sperm quality study, using lower than normal sperm doses to detect small differences between groups (Ward et al. 2003 Theriogenology 59, 1575). Frozen semen was available from 8 Holstein bulls kept outdoors in northern Spain, collected during winter, spring and summer. Bovine ovaries, Holstein and Swedish Red breeds, were obtained from an abattoir in spring. Oocytes were matured and fertilized in vitro with a low dose of frozen-thawed sperm (sperm:oocyte ratio 2500:1). After fertilization, presumptive embryos were evaluated at 44h for cleavage and on Day 8 for blastocyst development. Number of sperm binding to the zona pellucida and number of nuclei in developing blastocysts were assessed after staining with Hoechst 33342. There were 2 or 3 replicates per bull. For 555 oocytes inseminated, cleavage rates for winter, spring and summer semen collections were 42, 49 and 47%, respectively; blastocyst rates were 7, 12 and 8%; blastocyst cell numbers were 8.7, 10.2 and 7.8; and mean number of sperm bound was 0.70, 0.63 and 0.50. Although there were no differences (P>0.05) due to season of semen collection for any of these end points, individual bulls had considerable variation in cleavage rate. In conclusion, despite previously published differences in sperm quality with season from these bulls, ability of frozen-thawed sperm to fertilize bovine oocytes and initial embryo development in vitro were not affected by season of semen collection, at least for oocytes collected in spring. However, bull-to-bull variation in cleavage rate was high.
The authors gratefully acknowledge the financial support of the Ministerio de Economía y Competitividad and FEDER (AGL2016-79802-P). M. Sabes-Alsina was supported by a PIF from the Universitat Autònoma de Barcelona and by the STS of the Epiconcept COST Action; J. M. Morrell was funded by the Swedish Research Council for the environment, agricultural sciences and spatial planning (FORMAS; 221-2010-1241) and the Swedish Farmers’ Association (SLF; 1330039).
153 Sperm metabolomic landscape associated with bull fertility
Authors: Menezes E. , Santos F. , Velho A. , Dinh T. , Kaya A. , Topper E. , Didion B. , Moura A. , Memili E.
Published: December 2018

Sub-fertility fertility in bulls decreases the efficiency and profitability of cattle production because AI allows a single bull to inseminate thousands of cows. In recent decades, there has been a general decline in fertility of bulls, even among those exhibiting normal sperm motility and morphology. Despite advances in technology and knowledge, molecular, cellular and physiological mechanisms underlying the causes of low fertility in bulls are currently unclear. Therefore, the objective of this study was to identify sperm metabolites associated with fertility in Holstein bulls. The metabolome of sperm from 10 mature bulls with high fertility (HF, n=5) and low fertility (LF, n=5) was identified by gas chromatography coupled to mass spectrometry. Bull fertility was based on the sire conception rates deviating from the average. Statistical analysis was performed by using MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/). A total of 22 metabolites were detected and categorized according to their major chemical classes, including amino acids, peptides/analogues, carbohydrates/carbohydrate conjugates, fatty acids, steroids/steroid derivatives, keto acids and derivatives, carboxylic acids, and other organic and inorganic compounds. Organic acids and derivatives as well as fatty acids were the major compounds in bull spermatozoa. Seven organic acids and derivatives were detected, including benzoic acid, carbonate, carbamate dimethyl, carbamate trimethyl, lactic acid, oxalic acid, and urea. Five fatty acids were identified including oleic acid, oleanitrile, nonanoic acid, and palmitic acid. Oleic acid, phosphoric acid, phosphine, carbamate trimethyl, and glycerol were the most abundant metabolites in bull sperm, whereas benzoic acid, acetic acid, l-serine, carbamate, and 2-ketobutyric acid were the least predominant metabolites present in bull sperm. Multivariate analysis (partial least squares-discriminant analysis) of the sperm metabolome showed a clear separation between HF and LF bulls. Variable importance in projection (VIP) score demonstrated that metabolites with VIP >1.5 were gamma-aminobutyric acid (VIP=2.01), carbamate trimethyl (VIP=1.88), benzoic acid (VIP=1.86), and lactic acid (VIP=1.81). Abundance ratios of gamma-aminobutyric acid, carbamate trimethyl, benzoic acid, and lactic acid was greater in HF bulls compared with LF animals. According to univariate analysis, abundance ratios of gamma-aminobutyric acid (P=0.03) and carbamate trimethyl (P=0.047) were greater in HF than in LF bulls. Gamma-aminobutyric acid was positively correlated with carbamate trimethyl (r=0.94; P<0.0001) and benzoic acid (r=0.74; P=0.0139). Benzoic acid was positively correlated with carbamate trimethyl (r=0.75; P=0.0107) and carbamate dimethyl (r=0.68; P=0.0274). The identified metabolites can serve as potential markers to evaluate semen quality and predict bull fertility.
155 Whole genome association analysis suggests an influence of inbreeding on bull sperm morphometry
Authors: Azcona F. , Sole M. , Dorado J. , Ross P. , Terán E. , Demyda-Peyrás S.
Published: December 2018

Inbreeding depression, the genetic condition caused by mating related individuals, is particularly important in several cattle breeds with limited effective populations. This condition is often associated with decreases in performance, including fertility. Furthermore, sperm head morphometry was associated with fertility in several species. To our knowledge, the influence of inbreeding on sperm morphometry has not been widely reported in cattle. In this study, a Sperm Class Analyzer (SCA™, Microptic S.L., Barcelona, Spain) was used to characterise 7 sperm morphometry parameters in 59 Retinta bulls, including sperm head length, width, perimeter, ellipticity, elongation, regularity and rugosity. Two replicates of at least 100 sperm heads, from 2 frozen semen samples, were assessed per individual (n=200 sperm per bull). Additionally, all individuals were genotyped with the Axiom Bos1 high density SNP Array (Affymetrix, Santa Clara, CA, USA). The molecular-based inbreeding factor (FROH; mean 12.5%, range 1.75 to 33.0) had very low correlations with all sperm morphometry parameters. On average, sperm heads from bulls with higher FROH had a smaller area, but variability was high. Correlations between inbreeding and sperm shape were low and negative, length (r=−0.1449; P<0.01), width (r=−0.2494; P<0.01), and rugosity (r=−0.1086; P>0.01) being the most informative. Whole-genome association study (GWAS) analysis, performed using molecular inbreeding as co-factor, revealed 8 SNP, located on chromosomes BTA2, BTA5, BTA7, and BTA11, significantly associated with sperm head regularity and rugosity. Genomic analysis revealed that genes SLF1 and TMEFF2 are located close to enriched SNP. Gene SLF1 (SMC5-SMC6 complex localization factor 1) is associated with the regulation of protein complex and cytoskeleton assembly, whereas TMEFF2 (transmembrane protein with EGF like and 2 follistatin like domains 2) is associated with integral components of cell membrane and cell spreading and development. Therefore, we inferred that SLF1 and TMEFF2 may be involved in variations of sperm head shape. In this preliminary study, there was evidence of a potential influence of inbreeding on sperm morphometry in a beef cattle breed. However, additional studies, ideally including more individuals and additional breeds, are necessary to determine the validity of this potential association.
156 Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation
Authors: Felmer R. , Arroyo-Salvo C. , Fuentes F. , Cabrera P. , Treulen F. , Silva M. , Arias M. E.
Published: December 2018

Conventional IVF has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. Thus, the first part of this study aimed to compare HTF (Summers and Biggers, 2003 Hum. Reprod. Update9, 557-582, DOI: 10.1093/humupd/dmg039) and Whitten’s (McPartlin et al. 2009 Biol. Reprod.81, 199-206, DOI: 10.1095/biolreprod.108.074880) media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine on sperm motility parameters was evaluated in both media at different incubation times. Fresh semen from 3 Chilote stallions was collected, diluted to 10×106 sperm mL−1 in capacitating (7mg mL−1 BSA and 25mM NaHCO3) and non-capacitating (without BSA and NaHCO3) HTF and Whitten’s media and incubated for 30 and 120min at 38°C in air atmosphere. Integrity and destabilisation of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester perchlorate, acrosome membrane integrity by peanut agglutinin/fluorescein isothiocyanate and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® in a FACSCanto II flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). A total of 10,000 sperm events were acquired from each measurement (n=3 replicates for each stallion). Motility parameters were evaluated using the integrated semen analysis system (ISAS®, Selinion Medical, Brussels, Belgium). Percentage data were arcsine transformed and subjected to a 2-way ANOVA with Bonferroni’s post hoc test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). We found no differences between Whitten’s and HTF media in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30 and 120min of incubation. Membrane fluidity (MC540) increased in both media at 30 and 120min of incubation compared with non-capacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2 and 4h of incubation compared with non-capacitating conditions, without differences between media. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.
Funding support was received from FONDECYT 1160467 CONICYT, Chile.
16 False-positive rate after anticipation of early pregnancy diagnosis for resynchronization of ovulation in Bos indicus heifers
Authors: Simões L. M. S. , Lima E. A. , Santos A. P. C. , Orlandi R. E. , Bottino M. P. , Marinho P. H. A. , Scandiuzzi Junior L. A. , Massoneto J. P. M. , Souza A. H. , Baruselli P. S. , Sales J. N. S.
Published: December 2018

The objective was to determine the incidence of false-positive pregnancy diagnosis following the use of colour Doppler ultrasonography 20 and 22 days after fixed-time AI (FTAI) in Bos indicus heifers submitted to resynchronization 14 days after first FTAI. In the study, 512 Nellore heifers at 24.5±0.8 months of age and body condition score of 3.4±0.1 were used beginning 14 days after FTAI. On Day 14, heifers received 50mg of short-acting progesterone (Afisterone®, CEVA, São Paulo, Brazil) and a progesterone device (Prociclar®, CEVA). Eight days later (Day 22), the progesterone device was removed. A duplex B-mode (grayscale) and pulse-wave colour Doppler ultrasound instrument (M5, Mindray, Shenzhen, China) equipped with a multifrequency linear transducer was used for the examination of luteal blood flow on Days 20 and 22. The percentage of luteal area with colour Doppler signals of blood flow at each examination was determined as previously described (Ginther 2007) and was classified as low [corpus luteum (CL) with less than 25% vascularized area], intermediate (CL with 25-75% vascularized area), or high (CL with more than 75% vascularized area). Heifers with intermediate and high luteal blood flow were diagnosed as pregnant by Doppler ultrasonography, and heifers with low luteal blood flow were diagnosed as nonpregnant. Heifers diagnosed as pregnant by Doppler ultrasonography were examined 30 days after FTAI by B-mode ultrasound examination for pregnancy diagnosis and to determine the number of false positives (heifers pregnant by Doppler ultrasonography and nonpregnant by B-mode ultrasonography). Statistical analysis was performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Pregnancy rate by Doppler ultrasonography [Day 20=60.5% (310/512), Day 22=55.3% (283/512); P=0.10] and false-positive percentage [Day 20=29.7% (92/310), Day 22=23.0% (65/2830); P=0.06] were similar on pregnancy diagnoses by Doppler ultrasonography performed 20 and 22 days after FTAI. Furthermore, in the period from 20 to 22 days after FTAI, luteolysis was verified in 5.8% (30/512) of heifers. In conclusion, anticipation of pregnancy diagnosis in 2 days by evaluation of luteal blood flow with colour Doppler ultrasonography (20 days after FTAI) does not interfere with pregnancy rate by Doppler ultrasound and percentage of false positives in Bos indicus heifers.
160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation
Authors: Wani N. A. , Hong S.-B.
Published: December 2018

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P<0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.
162 Developmental competence of bovine oocytes matured in defined medium
Authors: Helland C. M. , Reichelderfer R. L. , Owen C. M. , Barcelo-Fimbres M. , Altermatt J. L. , Campos-Chillon L. F.
Published: December 2018

Routinely, bovine oocytes are matured in medium supplemented with 10% fetal bovine serum (FBS). However, due to the undefined nature of FBS and biosafety concerns, a standardized protein supplementation alternative is needed. We hypothesised that oocytes matured in defined medium supplemented with 5% serum substitute (DM) would have higher developmental competence than those matured in FBS-supplemented medium (Control). In 6 replicates, in vitro-produced embryos were produced during the summer season by aspirating cumulus-oocyte complexes from 2- to 8-mm follicles of abattoir ovaries (LAB-COC), or aspirated from FSH-stimulated Holstein and Jersey donors (OPU-COC, n=20; 53 sessions). The LAB-COC (n=1307) and OPU-COC (n=679) were matured for 23h in control medium or DM supplemented with pyruvate and gonadotropins at 6% CO2 in air, fertilized with semen from 1 of 3 or 15 bulls, respectively, and cultured in SCF2 at 38.5°C, 6% CO2, 5% O2 and 89% N2. On Day 7.5 post-fertilization, blastocyst rates were evaluated, and embryos (n=4/replicate) from each group of LAB-COC were stained with 1µg mL−1 Nile Red for lipid quantification, 5 µM Cell Rox® Green (Thermo Fisher Scientific, Waltham, MA, USA) for reactive oxygen species (ROS) assessment and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA) adjusted per cell number. Data were analysed by one-way ANOVA using SAS software (SAS Institute Inc., Cary, NC, USA). Results (Table 1) indicate similar cleavage and blastocyst rates and mitochondrial polarity for embryos from LAB-COC in Control v. DM (P>0.05). However, ROS and lipid content were higher in Control than in DM (P<0.05). For OPU-COC, DM resulted in similar cleavage and blastocyst rates and embryos produced per OPU session compared with Control (P>0.05). Results suggest that maturing oocytes (LAB and OPU) in DM maintain developmental competence, and the resulting embryos had lower lipid content and ROS than those matured in Control medium. Cryopreservation studies and evaluation of pregnancy rates are ongoing.

Table 1.Developmental competence of oocytes matured in control or defined medium with 5% serum substitute (DM)
165 In vitro maturation of ovine and caprine oocytes during breeding and nonbreeding seasons
Authors: Markle M. , Mak C. K. , Medina V. , Pinto C. R. F.
Published: December 2018

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P<0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.
167 Basal and maximal oxygen consumption of oocytes from young and old mares
Authors: Catandi G. , Obeidat Y. , Chicco A. , Chen T. , Carnevale E.
Published: December 2018

Mitochondria play a critical role in oocyte developmental competence by providing energy to the oocyte through oxidative phosphorylation. We hypothesised that oxygen consumption, a measure of mitochondrial function, would be lower in the oocytes of old versus young mares as a potential mechanism of reduced developmental competence associated with equine aging. To test this hypothesis, we used a novel electrochemical sensor device to compare basal and maximal oxygen consumption rates of oocytes obtained from mares aged 6 to 12 years (Young, n=8 from 7 mares) and=20 years (Old, n=12 from 8 mares). Cumulus-oocyte complexes (COC) were collected by transvaginal, ultrasound-guided follicular aspirations of dominant follicles (35mm) during oestrus and at 16±2h after induction of follicular maturation (2000IU of hCG and 0.75mg of deslorelin acetate). Recovered COC were incubated in medium [TCM-199; Life Technologies, Grand Island, NY, USA) with 10% fetal calf serum, 25mg mL−1 gentamicin, and 0.2mM pyruvate] at 38.5°C in 5% CO2 and air for 26±2h. After maturation, COC were stripped of cumulus cells and checked for the presence of an extruded polar body before electrochemical measurements using amperometry. A microchamber with a 3-electrode system (working, counter, and reference electrodes) was filled with 120µL of medium (G-MOPS™, Vitrolife, Englewood, CO, USA) and overlaid with 120µL of paraffin oil (OVOIL™, Vitrolife). The baseline current was measured before adding the oocyte through the oil layer and over the centre of the working electrode. Oxygen consumption was monitored as the linear decrease in the oxygen reduction current over time, which was converted to femtomoles of O2 consumed per second (fmol s−1) using a calibration curve for the sensor. Basal (nonstimulated) oxygen consumption was measured for 5min, followed by assessment of maximal noncoupled respiratory capacity obtained by titrating 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 8-min intervals. The highest value observed during CCCP titrations was considered the maximal oxygen consumption. Two-tailed Student’s t-tests were used to analyse data. Oocytes from young mares had higher basal oxygen consumption compared with old mares, respectively (mean±s.e.m., 1.74±0.18 and 1.27±0.09 fmol s−1; P=0.04) as well as higher maximal oxygen consumption (2.67±0.19 and 2.05±0.12 fmol s−1; P=0.016). Mitochondrial efficiency (maximal/basal oxygen consumption) was not different for Young and Old, respectively (1.62±0.13 and 1.65±0.07; P=0.9). In summary, we used a novel electrochemical sensor device to measure basal and maximal oxygen consumption of oocytes and to confirm our hypothesis that aging impairs mitochondrial respiratory capacity in equine oocytes, which may contribute to age-associated changes in equine fertility.
169 Use of stored zonae pellucidae from young and old mares to study sperm–oocyte binding
Authors: Stilz C. R. , Stoltzfus M. , Boyd B. , Bresnahan D. R.
Published: December 2018

Natural and standard IVF requires sperm binding to the zona pellucida (ZP); however, difficulties in obtaining equine ZP limit research in this area. In a previous study, an in vitro assay was used to assess ZP sperm binding for oocytes collected from old and young mares’ dominant follicles, with similar numbers of sperm bound per ZP (means of 45.5 and 58.1 sperm per ZP; P=0.12) (Lupole et al. 2010 Anim. Reprod. Sci. 121, 252-253). We hypothesised that ZP from uncleaved equine oocytes after intracytoplasmic sperm injection (ICSI) could be used to study ZP sperm binding. For this study, oocytes from mares >20 years (old, n=13 oocytes) and 4 to 12 years (young, n=16 oocytes) were collected by transvaginal, ultrasound-guided aspirations and injected with a single sperm. Only oocytes that failed to cleave into at least 2 cells within 2 days after ICSI were used. The oocytes were stored in hyperosmotic salt solution at 4°C for 2 years. The sperm binding assay was performed similar to the previous study (Lupole et al. 2010). Oocytes were removed from salt solution and placed into TALP and incubated for 1h at 38.5°C in 6% CO2 and air. Following incubation, oocytes were moved to a 45-µL drop of TALP. Frozen-thawed sperm (100,000 total sperm) from one stallion of good fertility were added and incubated for 2h. Oocytes were then washed through 4 drops of TALP using a pipette tip with a 175-µm inner diameter to remove loosely bound sperm. Oocytes and tightly bound sperm were stained in 4’6-diamidino-2-phenyindole (DAPI; 10µg mL−1) for 15min. The oocytes were secured on a glass slide under a coverslip that was supported by a mix of paraffin and petroleum jelly and viewed at 400× magnification using an epifluorescence microscope (Nikon Eclipse E800, Nikon, Tokyo, Japan). The number of tightly bound sperm was recorded. Sperm counts for age groups were compared using Student’s t-test, with data presented as mean±s.e.m. Sperm binding was not different between groups (59.2±16.9 for old mares and 36.8±10.6 for young mares; P=0.25). As previously observed with oocytes that had not been injected with sperm and minimal time in salt storage, we found no significant effect of mare age on sperm binding to the ZP. In addition, numbers of sperm bound to the ZP were similar between projects. Our findings suggest that equine oocytes that fail to cleave after ICSI could provide a valuable source of ZP for research. Further work is ongoing to determine the effect of time that oocytes are stored in salt solution on sperm binding.
170 The effects of linoleic and linolenic acid supplementation on the in vitro maturation of pig oocytes in a heat-stressed environment
Authors: Mentler M. , Current J. , Whitaker B.
Published: December 2018

Elevated environmental temperatures induce heat stress, which can cause a depression in fertility and early embryonic development. Fatty acids initiate an endergonic reaction that is able to absorb cellular heat, causing a decrease in intracellular temperature. Supplementing linoleic and linolenic acids to the maturation medium of pig oocytes at elevated temperatures reduces the effects of heat stress-induced damage during fertilization and embryonic development. However, the mechanism of action of fatty acids during oocyte maturation is unknown. Therefore, the objective of this study was to minimize heat stress-induced damage and characterise the intracellular oocyte mechanisms. Oocyte maturation media was supplemented with linoleic and linolenic acid during oocyte maturation at either 38.5 or 41.5°C. Oocytes (n=3094, r=5) were supplemented with 50μM linoleic acid, 50μM linolenic acid, 25μM of both, or 50μM of both during 40 to 44h of maturation and then evaluated for the formation of reactive oxygen species (n=239), intracellular glutathione concentrations (n=1005), glutathione peroxidase (n=1005), catalase (n=987), and superoxide dismutase (n=863) activities. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. There were no significant differences between the treatment groups matured at 38.5°C when comparing reactive oxygen species generation. Supplementation of linoleic or linolenic acid significantly decreased (P<0.05) reactive oxygen species generation in oocytes matured at 41.5°C compared with no supplementation at the same temperature. Supplementation of linoleic or linolenic acid or both significantly increased (P<0.05) intracellular glutathione concentrations compared with no supplementation at 38.5°C (23.37±1.23 pmol/oocyte) and 41.5°C (10.42±1.01 pmol/oocyte). There were no significant differences between the treatment groups matured at 38.5°C or 41.5°C when comparing glutathione peroxidase activity. Supplementation of linoleic or linolenic acid or both significantly increased (P<0.05) catalase and superoxide dismutase activities compared with no supplementation at 38.5°C and at 41.5°C. Superoxide dismutase activity was significantly higher (P<0.05) in oocytes matured at 41.5°C compared with those matured at 38.5°C. These results indicate that supplementing linoleic and linolenic acid to the maturation medium of pig oocytes at an elevated temperature reduces the effects of heat stress-induced damage by increasing intracellular glutathione concentrations and catalase and superoxide dismutase activities.
173 Assessment of the first polar body quality and viability in bovine
Authors: Briski O. , Duque-Rodríguez M. , Gambini A. , Leopardo N. P. , Ynsaurralde E. A. , Rodríguez M. B. , Bevacqua R. J. , Salamone D. F.
Published: December 2018

In female gametes, after the first asymmetric meiotic division, a mature oocyte in metaphase II and a first polar body (PB1) are generated. The PB1 contains one of each pair of homologous chromosomes present in the mature oocyte and its DNA can be used for preconception genetic diagnosis. The PB1 degenerates shortly after extrusion, possibly due to an apoptotic process; however, it has not yet been elucidated in bovine. Therefore, the objective of this study was to evaluate PB1 morphology changes, plasma membrane integrity, and the presence of DNA fragmentation during in vitro maturation (IVM). To this aim, cumulus-oocyte complexes collected from slaughterhouse ovaries were cultured in maturation medium in different groups according to IVM time: 16, 18, 20, 22, 24, and 26h. The PB1 were classified into 5 categories according to their morphology: grade (G)1, round PB1 with intact smooth membrane; G2, round or ovoid PB1 with intact membrane; G3, broken PB1 with a small PB1 fragment; G4, broken PB1 with a big PB1 fragment; and G5, completely damaged PB1. Grades 1 and 2 were considered good quality. Plasma membrane integrity was assessed by propidium iodide (PI) DNA staining; PI is a fluorescent intercalating agent that cannot cross the membrane of live cells. The presence of DNA fragmentation was detected at 16, 22, and 26h by TUNEL assay. Data were analysed by two-way ANOVA and Bonferroni post hoc test using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA) and differences were considered significant at P < 0.05. Our results (mean%±s.e.m.) showed that significantly more oocytes assessed at 18, 20, 22, and 24h after onset of IVM presented high-quality (G1) PB1 (18 h: 61.5±13.4%, 20 h: 73.5±9.6%, 22 h: 61.0±9.5%, 24 h: 60±5.1%), compared with those assessed at 16 and 26h (43.0±4.7%, 22.3±3.4%, respectively). The percentage of G2 PB1 did not change throughout the period studied (16 h: 34.0±13.5%, 18 h: 29.9±14.1%, 20 h: 22.0±7.1%, 22 h: 26.5±4.2%, 24 h: 23.3±4.9%, 26 h: 21.33±9.9%), but was significantly lower than that of G1 PB1 at 20, 22, and 24h. The proportion of damaged (G5) PB1 started to increase at 24h (14.3±8.6%), being highest at 26h (30.0±10.5%), in parallel with positive PI staining (P<0.05). Moreover, there was a significant increase of PB1 with DNA fragmentation at 26h (82.0±18.0%) compared with 16h (13.9±9.0%) and 22h (2.5±2.5%). Altogether, these findings demonstrate that PB1 remains stable and of good quality between 18 and 24 h; however, after this time, plasma membrane integrity is compromised and the DNA is fragmented, suggesting the occurrence of an apoptotic process. Our results could be helpful to determine the optimal time for using PB1 as a potential donor of genetic material.