Effects of N‐acetyl‐cysteine and N‐acetyl‐cysteine‐amide Supplementation on In Vitro Matured Porcine Oocytes

This study was conducted to evaluate the effects of different concentrations of the antioxidant N‐acetyl‐cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC‐amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm ) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post‐fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development.     

Beneficial Effect of Melatonin on Blastocyst In Vitro Production from Heat‐Stressed Bovine Oocytes

Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10^?12, 10^?9, 10^?4 m ; Experiment 4: 0, 10^?3 m ), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat‐stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10^?4 m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non‐HSO without melatonin. Melatonin did not have any effect in embryos from non‐HSO groups compared with the control. In Experiment 4, 10^?3 m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non‐HSO when compared to melatonin‐untreated oocytes/embryos. In conclusion, 10^?4 m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.     

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co‐culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co‐cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non‐co‐cultured somatic cell nuclear transfer (SCNT‐DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT‐DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation‐related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT‐DO group. Similarly, while the mRNA levels of the deacetylation‐related genes HDAC2 and HDAC3 were significantly higher in the SCNT‐DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co‐culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.     

Differential Expression of IGF Family Members in Heat‐Stressed Embryos Produced In Vitro from OPU‐Derived Oocytes of Nelore (Bos indicus) and Holstein (Bos taurus) Cows

The IGF system is related to embryo quality. We aim to determine the effect of the heat stress on the mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4, and PAPPA in in vitro production (IVP) blastocysts from Nelore and Holstein after ovum pick up (OPU) to better understand the differences between these breeds. Oocytes from four Nelore and seven Holstein were collected in six OPU sessions. Following in vitro maturation and fertilization using six Nelore or Holstein sires, embryos were divided into control (cultured at 39°C) and heat stress (HS; exposed to 41°C for 9 h). Blastocysts were submitted to RNA extraction. The IGF1 expression was higher in blastocysts under HS in both breeds, and the expression of IGFBP2 and IGFBP4 was higher in Holstein blastocysts under HS. The high PAPPA expression and the low expression of IGFBP2 and IGFBP4 are associated with a more efficient degradation of IGFBPs, which results in greater IGF bioavailability in Nelore blastocysts and may contribute to the superior HS tolerance in Nelore, when compared to Holstein.     

Apoptosis‐Like Events and In Vitro Fertilization Capacity of Sex‐Sorted Bovine Sperm

This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC‐1) and TUNEL) for flow cytometric analysis of apoptosis in sex‐sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (?ψm) showed that the sex‐sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex‐sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex‐sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex‐sorted bovine sperm.     

The In Vitro Development of Bovine Oocytes after Maturation in Glucose and β-Hydroxybutyrate Concentrations Associated with Negative Energy Balance in Dairy Cows

Negative energy balance (NEB) in high yielding dairy cows early postpartum may affect oocyte quality. Therefore, we tested the effect of two different beta-hydroxybutyrate (BHB) and glucose concentrations, which are associated with subclinical or clinical ketosis, during in vitro maturation (IVM) on the developmental competence of bovine oocytes. In Expt 1, subclinical ketosis conditions were imitated. Oocytes were matured in four different serum-free media with two glucose concentrations (g1=2.75 mm or G1=5.5 mm glucose) and with or without BHB (BHB1=1.8 mm BHB). Following maturation groups were used: g1, G1, g1:BHB1 and G1:BHB1. In Expt 2, clinical ketosis conditions were mimicked by using the concentrations: g2=1.375 mm or G2=3.1 mm glucose and BHB2=4.0 mm BHB. The combinations used were: g2, G2, g2:BHB2 and G2:BHB2. After IVM and in vitro fertilization (IVF), presumptive zygotes were routinely cultured for 7 days in synthetic oviduct fluid [SOF; 5% fetal calf serum (FCS)]. At 48 h and 8 days pi, cleavage rate and number of blastocysts were recorded respectively. The results demonstrated that the maturation conditions mimicking subclinical (g1:BHB1) and clinical ketosis (g2 : BHB2) resulted in an impaired developmental competence of the oocyte after maturation. Especially the moderately low (g1) or extremely low glucose (g2) concentrations were responsible for this detrimental effect that was associated with a blocked cumulus expansion. Only in moderately low glucose conditions (g1:BHB1), BHB exerted an additive toxic effect during oocyte maturation resulting in a reduced blastocyst rate. Conclusively, our results may suggest that subclinical and clinical ketosis can affect the oocyte's developmental competence most likely through a directly adverse effect of the low glucose concentrations on oocyte maturation. Only in subclinical conditions this harmful effect may be aggravated by BHB.     

Post‐Warming Competence of In Vivo Matured Rabbit Oocytes Treated with Cytoskeletal Stabilization (Taxol) and Cytoskeletal Relaxant (Cytochalasin B) Before Vitrification

The aim of this study was to investigate the effect of Taxol and Cytochalasin B on the spindle, chromosome configuration and development to blastocyst stage after parthenogenesis activation of in vivo matured rabbit oocytes after vitrification. Oocytes were randomized into four groups: oocytes treated with Cytochalasin B or Taxol before vitrification, oocytes without treatment before vitrification and fresh oocytes. Oocytes were vitrified using Cryotop method, and meiotic spindle and chromosomal distribution were assessed with a confocal laser scanning microscopy. To determine oocyte competence, in vitro development of oocytes was assessed with parthenogenesis activation. There were no significant differences in the frequencies of normal spindle (33.0%, 31.0% and 32.6%, for non‐treated, Taxol‐treated and Cytochalasin B‐treated oocytes, respectively) and chromosome (48.3%, 46.6% and 34.8%, for non‐treated, Taxol‐treated oocytes and Cytochalasin B‐treated oocytes respectively) in vitrified groups, but significantly lower than those of fresh group (89.7% and 90.2%, for normal spindle and chromosome organization, respectively). No statistical differences were found in the cleavage and blastocyst development rates between non‐treated and Taxol‐treated oocytes (7.7% and 1.5% and 13.7% and 4.6%, for non‐treated and Taxol‐treated oocytes, respectively), although they were significantly lower than in the fresh group (42.3% and 32.1%, for cleavage and blastocyst development, respectively). Oocytes treated with Cytochalasin B failed to reach blastocyst stage. Normal spindle, chromosome configuration and blastocyst development of in vivo matured rabbit oocytes were damaged in vitrification, which was not improved by Taxol and Cytochalasin B pre‐treatment before vitrification. Moreover, a detrimental effect on blastocyst development of Cytochalasin B pre‐treatment before vitrification was observed.     

Dietary lysine requirement for 7–16 kg pigs fed wheat–corn–soybean meal‐based diets

Two experiments were conducted to determine the lysine requirement of weaned pigs [Duroc × (Yorkshire × Landrace)] with an average initial BW of 7 kg and fed wheat–corn–soybean meal‐based diets. The experiments were conducted for 21 days during which piglets had free access to diets and water. Average daily gain (ADG), average daily feed intake (ADFI) and gain to feed ratio (G:F) were determined on day 7, 14 and 21. Blood samples were collected on day 0 and 14 to determine plasma urea nitrogen (PUN) concentration. In experiment 1, 96 weaned pigs were housed four per pen and allocated to four dietary treatments with six replicates per treatment. The diets contained 0.99%, 1.23%, 1.51% and 1.81% standardized ileal digestible (SID) lysine, respectively, corrected analysed values. The rest of the AA were provided to meet the ideal AA ratio for protein accretion. Increasing dietary lysine content linearly increased (p < 0.05) ADG and G:F. In experiment 2, 90 piglets were housed three per pen and allocated to five dietary treatments with six replicates per treatment. The five diets contained 1.03%, 1.25%, 1.31%, 1.36% and 1.51% SID lysine, respectively, corrected analysed values. Increasing dietary lysine content linearly increased (p < 0.05) G:F, linearly decreased (p < 0.05) day‐14 PUN and quadratically (p < 0.05) increased ADG and ADFI. The ADG data from experiment 2 were subjected to linear and quadratic broken‐lines regression analyses, and the SID lysine requirement was determined to be 1.29% and 1.34% respectively. On average, optimal dietary SID lysine content for optimal growth of 7–16 kg weaned piglets fed wheat–corn–SBM‐based diets was estimated to be 1.32%; at this level, the ADG and ADFI were 444 and 560 g, respectively, thus representing an SID lysine requirement, expressed on daily intake basis as, 7.4 g/day or 16.76 mg/g gain.     

In Vitro Biomechanical Comparison of a Modified 5.5 mm Locking Compression Plate Fixation with a 5.5 mm Locking Compression Plate Fixation of Osteotomized Equine Third Metacarpal Bones

Objectives: To compare number of cycles to failure for palmarodorsal 4‐point bending of a modified 5.5 mm broad locking compression plate (M5.5‐LCP) fixation with a 5.5 mm broad LCP (5.5‐LCP) fixation used to repair osteotomized equine third metacarpal (MC3) bones. Study Design: In vitro biomechanical testing. Animal Population: Adult equine cadaveric MC3 bones (n=6 pairs). Methods: An 8‐hole, M5.5‐LCP, obtained by having a 1.0 mm thickness removed from the bone contact portion of the 5.5‐LCP, was applied to the dorsal surface of 1 randomly selected MC3 from each pair, and an 8‐hole, 5.5‐LCP was applied dorsally to the contralateral bone from each pair using a combination of cortical and locking screws. Plates and screws were applied using standard ASIF techniques to MC3 bones with a mid‐diaphyseal osteotomy. MC3 constructs had palmarodorsal 4‐point bending cyclic fatigue testing. Mean cycles to failure for each method were compared using a paired t‐test within each group. Significance was set at P<.05. Results: Mean±SD cycles to failure of the M5.5‐LCP fixation (188,641±17,971) was significantly greater than that of the 5.5‐LCP fixation (166,497±15,539). Conclusion: M5.5‐LCP fixation was superior to 5.5‐LCP fixation of osteotomized equine MC3 bones in resisting cyclic fatigue under palmarodorsal 4‐point bending. Clinical Relevance: This suggests that biological plate fixation is not the ideal choice for osteotomized equine MC3 bones.     

An In Vitro Biomechanical Comparison of a 5.5 mm Locking Compression Plate Fixation with a 4.5 mm Locking Compression Plate Fixation of Osteotomized Equine Third Metacarpal Bones

Objectives: To compare the monotonic biomechanical properties and fatigue life of a 5.5‐mm‐broad locking compression plate (5.5 LCP) fixation with a 4.5‐mm‐broad locking compression plate (4.5 LCP) fixation to repair osteotomized equine 3rd metacarpal (MC3) bones. Study Design: In vitro biomechanical testing of paired cadaveric equine MC3 with a middiaphyseal osteotomy, stabilized by 1 of 2 methods for fracture fixation. Animal Population: Fifteen pairs of adult equine cadaveric MC3 bones. Methods: Fifteen pairs of equine MC3 were divided into 3 test groups (5 pairs each) for (1) 4‐point bending single cycle to failure testing, (2) 4‐point bending cyclic fatigue testing, and (3) torsional single cycle to failure testing. An 8‐hole, 5.5 LCP was applied to the dorsal surface of 1 randomly selected bone from each pair and an 8‐hole, 4.5 LCP was applied dorsally to the contralateral bone from each pair using a combination of cortical and locking screws. All plates and screws were applied using standard ASIF techniques. All MC3 bones had middiaphyseal osteotomies. Mean test variable values for each method were compared using a paired t‐test within each group with significance set at P<.05. Results: Mean yield load, yield bending moment, composite rigidity, failure load, and failure bending moment, under 4‐point bending, single cycle to failure, of the 5.5 LCP fixation were significantly greater than those of the 4.5 LCP fixation. Mean cycles to failure in 4‐point bending of the 5.5 LCP fixation (170,535±19,166) was significantly greater than that of the 4.5 LCP fixation (129,629±14,054). Mean yield load, mean composite rigidity, and mean failure load under torsional testing, single cycle to failure was significantly greater for the broad 5.5 LCP fixation compared with the 4.5 LCP fixation. In single cycle to failure under torsion, the mean±SD values for the 5.5 LCP and the 4.5 LCP fixation techniques, respectively, were: yield load, 151.4±19.6 and 97.6±12.1 N m; composite rigidity, 790.3±58.1 and 412.3±28.1 N m/rad; and failure load: 162.1±20.2 and 117.9±14.6 N m. Conclusion: The 5.5 LCP was superior to the 4.5 LCP in resisting static overload forces (palmarodorsal 4‐point bending and torsional) and in resisting cyclic fatigue under palmarodorsal 4‐point bending. Clinical Relevance: These in vitro study results may provide information to aid in selection of an LCP for repair of equine long bone fractures.     

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm–Egg Binding,Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus‐associated protein, osteopontin (OPN), lipocalin‐type prostaglandin D synthase (L‐PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg‐associated OPN and L‐PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre‐incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L‐PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 10⁵ frozen‐thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre‐treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L‐PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.     

Developmental Competence and Embryo Quality of Small Oocytes from Pre‐pubertal Goats Cultured in IVM Medium Supplemented with Low Level of Hormones,Insulin–Transferrin–Selenium and Ascorbic Acid

The aim of this study was to test the effect of insulin–transferrin–selenium (ITS) and L‐ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre‐pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus–oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post‐warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre‐pubertal goat small oocytes in GM would be useful to improve the quality of in vitro‐produced blastocysts.     

Superovulatory Response of Zebu Cows Treated with pFSH in a Single Subcutaneous Injection Followed by an Additional Intramuscular Sub‐Dose 48 h Later

The aim of this study was to evaluate whether an additional intramuscular (im) injection of pFSH would increase the embryo production in zebu cows superovulated with a single subcutaneous (sc) pFSH injection. Twenty‐one Nelore cows were treated with a progesterone vaginal implant (Controlled Internal Drug Relased – CIDR B^®) and injected im with 2.5 mg estradiol benzoate. Four days later, cows were assigned randomly into three groups and superovulated with pFSH. Groups A and B received single sc injections of 400 and 320 IU, respectively; Group C received multiple im injections of 400 IU in decreasing doses at 12‐h intervals over 4 days. In the morning (07:00 am) of day 3 after starting superovulation, cows received im 150 mcg cloprostenol and Group B was additionally injected im with 80 IU of pFSH. CIDR‐B was withdrawn in the afternoon (07:00 pm). Cows were inseminated 48 and 62 h after the cloprostenol injection. Embryo collection and corpora lutea (CL) estimation were done 7 days after insemination. Alternation of treatments (crossover design) occurred at a 60‐day interval. There was no significant difference (p > 0.05) of CL counts among treatments. The total (transferable and no transferable) number of recovered embryos from Group A (6.9 ± 1.5) was not different from Group C (9.8 ± 1.2), whereas Group B (5.7 ± 1.5) was lower than Group C (p < 0.05). The number of transferable embryos from Groups A (2.4 ± 0.7) and B (1.7 ± 0.6) was lower (p < 0.05) than Group C (4.6 ± 1.2). Lesser (p < 0.05) embryo production from Group B was related to lower recovery rate (46.4%), compared with Groups A (65.1%) and C (81.7%). It was concluded that an additional im subdose of pFSH, injected 48 h after a single subcutaneous (sc) dose of pFSH, does not improve the embryo production in zebu cows.     

Long‐term prognosis of gastrojejunostomy in foals with gastric outflow obstruction: 16 cases (2001–2006)

Reasons for performing study: It has been suggested that the success of gastric bypass surgery in foals for the treatment of gastric outflow obstruction is poor. However, few reports exist evaluating the long‐term prognosis of these cases. Objectives: To determine the long‐term success of foals, including racing records, surgically treated for gastric outflow obstruction secondary to gastroduodenal ulceration. Methods: Medical records of foals undergoing surgical treatment of gastric outflow obstruction secondary to gastroduodenal ulceration were evaluated for clinical information. Owners, trainers and race records were evaluated regarding long‐term survival and racing success. Results and conclusions: Sixteen foals were included in the study, all treated with a gastrojejunostomy. All foals survived to immediate discharge from the hospital; 8 survived to racing age, with 7 of those entering training and 3 actually racing. Foals that did not survive to racing age had various post operative complications. The success rate for these foals appears somewhat better than that previously reported. Potential relevance: Gastrojejunostomy for the treatment of gastric outflow obstruction, secondary to gastric ulceration, is a valid treatment option for foals.     

Return to use and performance following exploratory celiotomy for colic in horses: 195 cases (2003–2010)

Reasons for performing study: There are few objective data on return to use and performance in horses following colic surgery. Objective: To investigate return to functional use of horses following colic surgery and factors associated with a negative outcome. Methods: The North Carolina State University Equine Colic Database was reviewed for horses that underwent exploratory celiotomy for colic (2003–2010). Horses were excluded from the study if they survived <6 months, had no intended use preoperatively, or if further data were not available at attempted follow‐up. Information retrieved included history, background, use, and selected pre‐, intra‐, and post operative factors. Telephone interviews were used to obtain follow‐up data. Logistic regression was used to investigate associations between clinical data and outcome, reported as odds ratios with a 95% confidence interval and corresponding P value. Results: Of patients surviving to 6 months, 133/195 (68%) were performing their intended use and 85/156 (54%) were at or above preoperative performance. At one year, 145/190 (76%) horses were performing their intended use and 101/153 (66%) were at or above preoperative performance. Animals were significantly less likely to return to use/performance if they had a previous celiotomy, stall rest for an orthopaedic condition, a nonstrangulating lesion type, incisional hernia, diarrhoea or laminitis. Conclusions: The overall prognosis for return to use and performance following colic surgery is fair to good. Multiple pre‐ and post operative factors may affect the likelihood of return to use and performance. Potential relevance: Targeted owner education regarding preoperative lameness, post operative rehabilitation and treatment for complications, such as incisional hernioplasty, may help inform owners about their horse's potential for return to use and performance following colic surgery.