Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus-oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 - 60%) and embryonic development rates to the blastocyst stage (10/48 - 21%) as the results obtained with the BCB control group oocytes (45/77 - 58% and 08/45 - 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB- oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB- (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB-; and (4) BCB+ matured in same IVM medium drop as (5) BCB- at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 - 44%) differed (P < 0.05) from the other groups [1 (145/241 - 60%); 2 (150/225 - 67%); 4 (201/318 - 63%) and 5 (21/33 - 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 - 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 - 21%; group 3: 17/102 - 17%; group 4: 46/201 - 23%; and group 5: 2/21 - 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB- oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB- oocyte group, BCB+ paracrine group and BCB- paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.

The influence of two oxygen tensions (5 and 20%) on fertilization, cleavage, development, and morphological quality of canine embryos produced in vitro was investigated. To assess embryo production, canine oocytes (N = 608) were matured in vitro at 20% oxygen tension in TCM 199 supplemented with glucose (11 mM) and 0.1% polyvinyl alcohol. Oocytes and sperm were coincubated at 37 °C for 24 hours in serum-free medium. Subsequently, presumptive zygotes were cultured in vitro in synthetic oviductal fluid in either 5% CO2 in air (20% oxygen; N = 298) or 5% O2, 5% CO2 and 90% N2 (5% oxygen; N = 310) for 7 days. Regardless of the oxygen concentration (5 vs. 20%), rates of pronucleus formation, cleavage, and embryo development up to the eight-cell stage did not differ (46/310 [14.8%] vs. 35/298 [11.7%], respectively; P > 0.05). Moreover, similar proportions of embryos developed in 20 and 5% oxygen tensions (18/298 vs. 27/310). The oocyte nuclear maturation (metaphase II), in terms of decondensed sperm heads, pronucleus formation, cleavage, and embryo development, was similar (P = 0.299) between the atmospheric (20% O2; 12.4% [37/298]) and reduced oxygen tensions (5% O2; 15.8% [49/310]) at all steps of the in vitro culture (IVC) after in vitro fertilization (IVF). To our knowledge, this was the first study that demonstrated that canine embryos can be produced using a low-oxygen in vitro culture system. Both oxygen tensions (5 and 20%) resulted in similar embryonic development and therefore were feasible for IVC of canine oocytes.

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The combined use of timed AI (TAI) and embryo transfer (TET) has the potential to increase reproductive efficiency in beef cattle. This study evaluated reproductive performance in beef cattle after TAI followed by TET of in vitro-produced embryos at the onset of the breeding season. A total of 476 multiparous non-suckling Bos taurus females (body condition scores of 2.9 ± 0.4 on 1 to 5 scale) were oestrous synchronized with 2 mg of oestradiol benzoate (IM) and a 1.9-g intravaginal progesterone release device (Day –11), which was removed on Day –2, followed by 0.48 mg of sodium cloprostenol, 400 IU of eCG, and 0.5 mg of oestradiol cypionate (IM). In experiment I [no heat detection (HD), or no HD, n = 387], TAI was carried out 48 h later (Day 0), whereas in experiment II (after HD, n = 89), AI was performed 12 h after the onset of oestrus up to 48 h after intravaginal insert removal, when remaining females were inseminated (Day 0). Day-7 blastocysts produced by IVF from abattoir-derived oocytes were individually transferred (TET) 7 days after TAI (Day 7) to 186/387 and 44/89 females in experiments I and II, respectively, ipsilateral to the corpus luteum. Then, fertile mature Bos taurus bulls were introduced on Day 12 into the herds (1:25) up to Day 90. Determinations of pregnancy outcome after TAI, TAI+TET or natural mating, twinning rates, and pregnancy losses were done by ultrasonography and rectal palpation on Days 30, 60, and 125. Data were analysed by the Chi-squared test (P < 0.05). Pregnancy rates (Day 30) were lower after TAI (104/201, 51.7%) than after TAI+TET (126/186, 67.7%) with no HD (experiment I), but similar between TAI (32/45, 71.1%) and TAI+TET (30/44, 68.2%) after HD (experiment II). Twinning rates were lower in TAI groups with no HD (6/104, 5.8%) and after HD (2/32, 6.2%) than in TAI+TET groups with either no HD (42/126, 33.3%) or with HD (14/30, 46.7%). Overall pregnancy was similar between groups after the end of the breeding season: 90.0% (181/201) and 90.3% (168/186) for TAI and TAI+TET with no HD, and 84.4% (38/45) and 84.1% (37/44) for TAI and TAI+TET after HD. Pregnancy losses were higher after TAI+TET with no HD (27/126, 21.4%) than TAI+TET after HD (3/30, 10.0%), and TAI with (2/32, 6.3%) or without (9/104, 8.7%) HD. The TAI+TET with no HD resulted in fewer fetuses per served (0.69) and pregnant (1.30) female than TAI+TET after HD (0.89 and 1.44), whereas TAI with no HD had fewer fetuses than TAI after HD per served (0.50 v. 0.69) but not per pregnant female (1.05 v. 1.03), with both being lower than the TAI+TET groups. In summary, TET after TAI with no HD increased pregnancy and twinning rates. Also, heat detection increased pregnancy rates after TAI and twinning rates after TAI+TET. The TAI+TET combination may be advisable for reproductive schemes with no HD, whereas no benefit of TAI+TET was seen over TAI regarding pregnancy rate if TAI is coupled with HD, but HD may increase prolificacy after TAI+TET. The economics of the use of TAI+TET is under evaluation, by assessing calving, weaning, and postnatal weight gain rates between groups.

Stress processes, such as hydrostatic pressure treatment of oocytes in different species, have been reported to increased embryo rate following in vitro maturation (IVM). However, studies on high gaseous pressure (HGP) pretreatment in IVM of oocytes from domestic animals are lacking in the literature. This experiment aimed to test HGP pretreatment of canine oocytes to increase meiosis achievement (metaphase II) after IVM. A total of 502 canine oocytes (6 replicates) were used in this study. Ovaries from 15 bitches were obtained from local shelters or rescue organizations after ovariohysterectomy. Sample collection was blind as to reproductive stage and dog age. The ovaries were transported to the laboratory in 0.9% NaCl and were processed within 3 h of collection. The ovarian cortex was sliced and washed in PBS with 1% FCS to release cumulus–oocyte complexes. Grade 1 and 2 cumulus–oocyte complexes were selected for IVM and randomly distributed into 3 treatment groups: HGP (oocytes placed in PBS and subjected to pressure chamber; 206 oocytes), ambient control (oocytes maintained in TCM-HEPES at room temperature for 60 min; 130 oocytes), and laboratory protocol (oocytes IVM after morphologic selection; 166 oocytes). The average pressure, initial and final temperature, and duration of oocytes in the HGP pretreatment were 76.19 atm (±0.92), 32.20°C (±5.17) and 27.71°C (±3.17), and 60 min, respectively. In vitro maturation was carried out for 72 h at 37°C in a high-glucose medium, consisting of TCM-199 with 2.2 mg mL–1 of sodium bicarbonate (11150, Gibco, Grand Island, NY, USA), and supplemented with 0.1% polyvinyl alcohol (P-8136, Sigma, St. Louis, MO, USA), 0.991 mg mL–1 of glucose (108337, Merck, Darmstadt, Germany), 50 µg mL–1 of gentamicin, 22 µg mL–1 of pyruvic acid, 20 µg mL–1 of oestradiol (E-8875, Sigma), 0.5 µg mL–1 of FSH (Folltropin-V, Vetepharm Inca), 0.03 IU mL–1 of hCG (Chorulon®, Intervet, Kenilworth, NJ, USA), under 20% oxygen tension. The number of oocytes at each stage (prophase to metaphase II) was recorded according to the morphology of nuclear content after staining with Hoechst 33342. For comparison purposes of nuclear maturation in oocytes, data were analysed by Fisher’s exact test. Differences at a P-value ≤0.05 were considered significant. Oocytes from the HGP, ambient control, and laboratory protocol groups had similar meiotic progression to the metaphase II stage (metaphase I–anaphase I–telophase I–metaphase II), and were 35.4% (73/206), 30.8% (40/130), and 34.9% (58/166), respectively (P ≥ 0.05). The proportion of oocytes without chromatin or having an irregular organisation was not different among groups. In conclusion, results indicate that HGP pretreatment as used in this experiment did not improve meiosis rates in IVM canine oocytes. Further investigations to understand the significance of HGP pretreatment in IVM and in vitro production of canine embryos are ongoing in our laboratory.