Generation of Buffalo (Bubalus bubalis) Transgenic Chimeric and Nuclear Transfer Embryos Using Embryonic Germ-Like Cells Expressing Enhanced Green Fluorescent Protein

The possibility of producing transgenic buffalo embryos by chimera and nuclear transfer (NT) using buffalo embryonic germ (EG)‐like cells expressing enhanced green fluorescent protein (EGFP) has been explored in this study. Buffalo EG‐like cells and fibroblasts with two to eight passages were transfected with the lined plasmid (pCE‐EGFP‐IRES‐Neo‐dNdB) using Lipofectamine^TM 2000 and selected by culturing in 200 μg/ml G418 for 6–8 days. G418 resistant fibroblasts and EG‐like cells were used for embryo chimera and NT. To produce blastocysts by chimera, 8–16 cells embryos were injected with EG‐like and fibroblast cells. Then, to produce blastocysts by NT, in vitro maturated oocytes were enucleated and afterwards EG‐like/fibroblast cells transferred into the perivitelline space. No statistical differences were observed for the total blastocyst produced by the chimeric method, using EG‐like and fibroblasts as donor cells, resulting on an accomplishment of 35.6% vs 33.3%, respectively. Nevertheless, besides from the 37 blastocysts produced, 23 (62.2%) from EG‐like cells expressed EGFP, none of blastocysts from foetal fibroblasts expressed this protein. When the NT method was used, no statistical difference among different generations was observed in the percentage of oocytes fused, cleaved, and developed to blastocysts after NT for EG‐like cells. On average, the percentage of oocytes fused, cleaved, and developed to blastocysts after NT was respectively 81.8%, 67.7% and 10.7%. For the expression of EGFP, from the 12 blastocysts produced by NT, 7 of them were positive, while none of NT embryos from EGFP positive fibroblasts developed to blastocysts. Results of the present study clearly demonstrated that gene transfected buffalo EG‐like cells have the ability to form chimeric embryos after injecting into buffalo early embryos and reprogramming ability after NT, which can be employed to produce transgenic buffalos through either embryo chimera or NT.     

Phosphate fractionation and spatial patterning in ancient ruins: A case study from Yucatan

As time passes, phosphorus (P) in soils tends to become more tightly bound with minerals. Phosphate fractionation enables the measurement of loosely versus tightly bound P. Archaeologists have used P fractionation as a chronometric technique: older soils should have greater proportions of P tightly bound with minerals. Research at Chunchucmil, a large Maya ruin in Yucatan, Mexico, has used extractable P concentrations and other lines of evidence to investigate ancient uses of space. Because contemporary Maya villagers use the land among the Chunchucmil ruins in a number of ways, and because the ancient land surfaces are neither sealed nor deeply buried, we used P fractionation to determine whether geochemical signatures of activities such as gardening resulted from ancient or modern inhabitants. The fractionation results at Chunchucmil are unusual in comparison to studies from other sites. Furthermore, there are strong correlations between the sum of P fractions and carbonate. We discuss potential explanations for these patterns in P fractionation data. These explanations consider local pedology, differential length of ancient occupation, and localized dumping of the byproducts of maize processing. To examine the chemical signatures of maize processing, specifically soaking maize in lime water, contemporary samples of sediments and residues from this activity were analyzed. Our results suggest that dumping water from maize soaking provides a potential explanation for the ancient P patterns.     

Treatment of anovulatory anoestrous postpartum dairy cows with a gonadotropin-releasing hormone (GnRH), prostaglandin F2𝛂, GnRH regimen or with progesterone and oestradiol benzoate

AIM: To compare 2 treatments for anovulatory anoestrus (AA) in postpartum dairy cows. The treatments were combinations of gonadotropin-releasing hormone (GnRH) and prostaglandin F_2𝛂 (PG) or progesterone (P4) and oestradiol benzoate (ODB).

METHODS: Forty AA cows from each of 5 herds were blocked by age (2 or >2 years old) and randomly assigned to 1 of 2 treatments. The first group (GPG) were treated with 250 𝛍g of a GnRH analogue, gonadorelin, followed 7 days later by 15 mg of the PG analogue, luprostiol. Two days later the cows were injected with 250 𝛍g of gonadorelin. Cows were artificially inseminated 16–24 h after the second GnRH injection. The second group (P4+ODB) were treated with an intravaginal P4 releasing device for 6 days, followed 24 h after device removal by injection of 1 mg of ODB. Cows were pregnancy tested 35–40 days after the initial insemination and twice again at 6–8 week intervals thereafter.

RESULTS: There was no significant difference between P4+ODB and GPG groups in the percentage of cows submitted for insemination in the first 7 days (94.0% vs 100% for P4+ODB vs GPG, respectively; p>0.3), in conception rate to first insemination within the first 7 days (43.6% vs 35.0% for P4+ODB vs GPG, respectively; p>0.2), in the percentage of cows conceiving in the first 28 days of the breeding period (68.0% vs 58.3%, P4+ODB vs GPG, respectively; p>0.1), or in median interval from the end of treatment to conception (20 vs 21days;p>0.1).

CONCLUSIONS: No differences in the reproductive performance of AA cows treated with either P4+ODB or GPG were detected. However, given the small number of animals enrolled, further data are required before the GPG protocol can be recommended for treatment of AA cows.     

Pregnancy loss in dairy cattle in the Waikato region of New Zealand

AIM: To define the incidence rate of pregnancy loss and risk factors for those losses in pasture-fed dairy cattle in the Waikato region of New Zealand.

METHODS: Cows (n=2,004) from 10 pasture-fed, spring-calving dairy herds in the Waikato were enrolled following confirmation of pregnancy 29–45 days after insemination, for inseminations that occurred within the first 16 days of the seasonal breeding period. Transrectal ultrasonographic examinations for pregnancy were conducted at approximately 6, 8, 10, 14 and 22 weeks gestation, and subsequent calving data were recovered. Pregnancy loss was defined as having occurred when a confirmed pregnancy was not rediagnosed, when gross abortion was detected, or when a cow calved <265 days after the confirmed conception date. Data were analysed using reverse stepwise logistic regression and Cox's proportional hazards analysis.

RESULTS: A total of 128 (6.4%) pregnancy losses were detected. The incidence rate was higher in early compared to late gestation (10.9 vs 2.8 losses/10,000 cow-days between Weeks 6–10 vs Weeks 10–14, respectively; p<0.001). Higher rates of loss were associated with the occurrence of clinical mastitis (Hazards ratio (HR)=1.57; p=0.071), being treated for anoestrus (HR=1.69; p=0.007), and in cows that had calving-to-conception intervals ≤63 days compared with those that had calving-to-conception intervals >92 days (HR=2.49; p=0.06). In addition, the rate of pregnancy loss differed between herds (p=0.05).

CONCLUSIONS: The highest rate of pregnancy loss occurred in early gestation. Clinical mastitis, anoestrus and calving late in the calving season were risk factors for pregnancy loss.

CLINICAL RELEVANCE: Pregnancy diagnosis using ultrasonography can be undertaken from 28 days post-insemination. However, due to the high rate of pregnancy loss at this stage of gestation, herdowners need to be warned of possible losses, and cows should be re-examined to confirm pregnancy before certification of pregnancy status is given.     

Effect of Ascorbic Acid Concentrations,Methods of Cooling and Freezing on Boer Goat Semen Cryopreservation

To improve the Boer goat semen quality during cryopreservation process, three experiments were carried out to investigate the effect of (i) different concentration of ascorbic acid supplementation (ii) rate of cooling with chilled semen characteristics and (iii) method of freezing on post‐thaw Boer goat sperm using Tris‐based extender. Ascorbic acid at 8.5 mg/ml improved the sperm parameters (motility, integrity of membrane and acrosome, morphology and viability), compared to control in cooled samples (p < 0.05). With regard to other concentrations and post‐thawed parameters, ascorbic acid at 2.5–8.5 mg/ml led to higher percentages of sperm motility and integrities of membrane and acrosome when compared to control (p < 0.05). Slow cooling rises to higher percentages of sperm motility, acrosome integrity and viability, in comparison with fast cooling, in terms of cooled and frozen samples (p < 0.05). Programmable freezing method produced the higher percentages of sperm motility, integrities of membrane and acrosome and viability when compared to the freezing method of polystyrene box during goat sperm freezing (p < 0.05). In conclusion, chilled and post‐thawed sperm quality of Boer goat was improved when a Tris‐based extender supplemented with ascorbic acid was used at stages of different cooling rates and freezing methods.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Influence of Superovulatory Protocols on In Vitro Production of Nellore (Bos indicus) Embryos

There are indications in the literature that delaying the period between ovarian superestimulation and ovum pick up (OPU) would induce follicles to a condition of initial atresia, which could be beneficial to oocyte development. In this work, we compared three protocols for OPU and in vitro production (IVP) of embryos, in Nellore cattle. Nellore cows (n = 18) were randomly allocated in three groups: Group 1 (OPU), Group 2 [Follicle stimulating hormone (FSH) and OPU] and Group 3 (FSH deprivation and OPU). Three OPUs were performed, and the animals were switched to a different group each time (crossover), in such a way that at the end of the experiment all cows received the 3 protocols. At random stage of the oestrous cycle (D‐2), all follicles ≥ 6 mm were aspirated to induce a new follicular wave 2 days afterwards (D0). In Group 1, OPU was performed on D2 and oocytes were processed to IVP. In Group 2, starting on D0, cows were superstimulated (FSH, Folltropin^®, 30 mg administered daily, i.m., during three consecutive days, total dose = 180 mg), and 6 h after the last FSH dose, they received exogenous luteinizing hormone (LH) (12.5 mg, i.m., Lutropin^®, D3). The OPU was performed 6 h after LH administration, i.e. 12 h after the last dose of FSH. Animals in Group 3 received the same treatment as those in Group 2, except that LH was administered 42 h after the last dose of FSH, and OPU occurred 6 h later. Therefore, in this group, follicles were deprived of FSH at 48 h. Both cleavage and blastocyst rates were similar (p > 0.05, anova ) among oocytes from Groups 1, 2 and 3, respectively: 77.4% (144/185) and 42.70% (79/185); 75.54% (105/139) and 31.65% (44/139); 63.52% (101/159) and 33.33% (53/159). However, hatched blastocyst rate was higher (p < 0.01) in Group 1 (30.27%, 56/185) when compared with Group 2 (11.51%, 16/139) or 3 (15.72%, 25/159). It is concluded that, contrary to previous work on European breeds (Bos taurus), ovarian superstimulation associated with deprivation of FSH and OPU (Group 3) did not increase IVP of Nellore embryos (Bos indicus). On the contrary, the highest hatched blastocyst rates were observed in oocytes from non‐superstimulated cows.