Qualitative and quantitative analysis of goat ovaries, follicles and oocytes in view of in vitro production of embryos

Goat ovaries were collected from the slaughterhouse and categorized as right, left, corpus luteum （CL）-present and -absent group and evaluated on the basis of weight （g）, length （cm）, width （cm）, number of follicles, follicles aspirated and number and state ofcumulus-oocyte-complexes （COCs）. Comparatively higher weight [（0.66±0.02） vs （0.64±0.02） g], length [（1.17±0.02） vs （1.1 ±0.02） cm] and width [（0.77±0.02） vs （0.74±0.02） cm] were found in right ovaries than those of left. On the other hand significantly （P〈0.05） higher weight [（0.71±0.03） vs （0.64±0.01） g] and width [（0.76±0.03） vs （0.75±0.01） cm] were found in CL-present group than those of CL-absent group of ovaries. The left ovaries contained comparatively higher number of normal COCs [（1.06±0.09） per ovary] than fight ovaries [（1.03±0.10） per ovary] and the similar trend was found in total number of follicles [（4.51±0.25） vs （4.30±0.23） per ovary] and follicles aspirated [（2.55±0.14） vs （2.52±0.12） per ovary]. But the total COCs per ovary was almost similar in both ovaries [right and left： （1.85±0.12） and （1.85±0.11） per ovary, respectively]. Higher number of total COCs [（1.87±0.09） vs （1.76±0.16） per ovary], total number of follicles [（4.45±0.19） vs （4.16±0.37） per ovary], follicles aspirated [（2.55±0.10） vs （2.48±0.21） per ovary] and normal COCs [（1.12±0.07） vs （0.76±0.14） per ovary] were found in CL-absent group than those of CL-present group of ovaries.

Reproductive fluids,used for the in vitro production of pig embryos,result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM

Background:In vitro embryo production(IVP)and embryo transfer(ET)are two very common assisted reproductive technologies(ART)in human and cattle.However,in pig,the combination of either procedures,or even their use separately,is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow.In addition,the potential impact of these two ART on the health of the offspring is unknown.We investigated here if the use of a modified IVP system,with natural reproductive fluids(RF)as supplements to the culture media,combined with a minimally invasive surgery to perform ET,affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results:The blastocyst rates obtained by both in vitro systems,conventional(C-IVP)and modified(RF-IVP),were similar.Pregnancy and farrowing rates were also similar.However,when compared to in vivo control(artificial insemination,AI),litter sizes of both IVP groups were lower,while placental efficiency was higher in AI than in RF-IVP.Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI,but not for RF-IVP group.Conclusions:The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

Effects of Stylosanthes scabra Forage Supplementation on in Vitro Gas Production and Fiber Degradation of Eragrostis Grass Hay

Natural pastures constitute a major component of ruminant livestock feed, and are the most cost-effective feed resource available for smallholder subsistence farmers. However, this feed resource does not meet animal nutritional requirement due to deficiency in nitrogen, energy and minerals. In addition, at maturity lignification is the major concern since it reduces digestibility and contributes to methane emission. Thus, the objective of this study was to evaluate the effect of supplementing low-quality Eragrostis grass hay with five (9281, 11,252, 11,255, 11,595 and 11,604) selected Stylosanthes scabra accessions on in vitro ruminal fermentation and neutral detergent fiber degradation. Therefore, in vitro study was conducted on grass hay, accessions and the mixture of grass hay with each accession included at two (15%, 30%) levels. The substrates (grass hay, accessions and the mixtures) were incubated in separate serum bottles for 72 h. Neutral detergent fiber (NDF) of the accessions ranged from 300 to 350 g/kg DM with crude protein (CP) value ranging from 177.5 to 184.1 g/kg DM. Eragrostis grass hay had NDF value of 813 g/kg DM, with CP value of 34.3 g/kg DM. Grass hay fermented slowly, it took 30 h for grass hay to produce gas volume above 50 mL, while Stylosanthes scabra accessions took 12 h. Supplementing grass hay with accessions significantly improved fermentation. However, it was observed that 15% inclusion took 30 h to produce gas volume above 50 mL, whereas at 30% inclusions it took 24 h for accession 9281, 11,595 and 11,604. Accession 11,604 improve grass fermentation by almost three times the value of grass hay in 2 h. Grass hay supplemented with accession 11,604 at 30% had a positive associative effect and significantly improved NDF degradability. In conclusion, accession 11,604 may be fed strategically as forage supplement to low-quality forage for ruminants.

Effect of Cadmium on Rat Leydig Cell Testosterone Production and DNA Integrity in vitro

Cadmium （Cd） is an elemental heavy metal with widely recognized toxicity. Its long-term use in industrial processes and daily activities has caused alarming levels of Cd contamination in the natural environment. According to the estimates by the Agency of Toxic Substances and Disease Registry in the US, 25 000 to 30 000 metric tons of Cd is annually released to the environment . Results of previous studies have demonstrated that several organs are targets of Cd, but the most important of these targeted organs may be the testes.

Rumen methane output and fermentation characteristics of gramineous forage and leguminous forage at differing harvest dates determined using an in vitro gas production technique

An in vitro rumen gas production technique was employed to determine the methane production and fermentation characteristics of Leymus chinensis and Medicago ruthenica at differing harvest dates（May 15,May 30,June 30,July 30,August 30 and September 30）,which are sequential phases within a single continuous growth of two 10-year-old pastures.To quantify the rate of degradation and compare in vitro rumen fermentation characteristic,a logistic-exponential model,where initial gas volume was zero（LE_0）,was used to fit gas production and methane output results.Dried,milled forage samples were incubated in vitro for 72 h at 39℃ and gas production was recorded intermittently throughout the incubation and gas samples were collected to measure methane production.Results showed that there were significant interactions between species and harvest for all chemical composition variables（P〈0.001） and condensed tannin content（P〈0.001）.L.chinensis produced more total gas and methane than M.ruthenica（P〈0.001）.Both total gas and methane production decreased lineally（P〈0.001） with advancing harvest date.The degradation rates of L.chinensis and M.ruthenica harvested on September 30 were lower than those on the other harvest dates（P〈0.01）.M.ruthenica fermented fluid had higher concentration of ammonia N（P〈0.05） and molar proportions of isobutyrate（P〈0.01）,valerate（P〈0.001） and isovalerate（P〈0.01） in total volatile fatty acids than L chinensis.Furthermore,concentration of isovalerate decreased cubically with advancing harvest date（P〈0.05）.In conclusion,M.ruthenica produced less methane than L.chinensis and the total gas and methane production decreased with advancing harvest date for both species,which may be due to the changes in contents of chemical compositions and condensed tannin in forages.

Influence of main dietary chemical constituents on the in vitro gas and methane production in diets for dairy cows

Background： Modification of chemical composition of diets fed to dairy cows might be a good strategy to reduce methane（CH4） production in the rumen. Notable reductions of CH4 production compared to conventional highroughages rations were more frequently observed for very concentrated diets or when fat supplements were used. In these cases, the reduction in the gas emission was mainly a consequence of an overall impairment of rumen function with a reduction of fiber digestibility. These strategies do not always comply with feeding standards used in intensive dairy farms and they are usually not applied owing to the risks of negative health and economic consequences.Thus, the present study evaluated the effects of seven commercial diets with contents of neutral detergent fiber（NDF）,protein and lipids ranging 325 to 435 g/kg DM, 115 to 194 g/kg DM, and 26 to 61 g/kg DM, respectively, on in vitro degradability, gas（GP）, and CH4 production.Results： In this experiment, changes in the dietary content of NDF, crude protein（CP） and lipids were always obtained at the expense or in favor of starch. A decreased of the dietary NDF content increased NDF（NDFd） and true DM（TDMd） degradability, and increased CH4 production per g of incubated DM（P 〈 0.001）, but not that per g of TDMd. An increase of the dietary CP level did not change in vitro NDFd and TDMd, decreased GP per g of incubated DM（P 〈 0.001）, but CH4 production per g of TDMd was not affected. An increased dietary lipid content reduced NDFd, TDMd,and GP per g of incubated DM, but it had no consequence on CH4 production per g of TDMd.Conclusions： It was concluded that, under commercial conditions, changes in dietary composition would produce small or negligible alterations of CH4 production per unit of TDMd, but greater differences in GP and CH4 production would be expected when these amounts are expressed per unit of DM intake. The use of TDMd as a standardizing parameter is proposed to account for possible difference in DM intake and productivity.

Methodological factors affecting gas and methane production during in vitro rumen fermentation evaluated by meta-analysis approach

Effects of some methodological factors on in vitro measures of gas production（GP, mL/g DM）, CH4production（mL/g DM） and proportion（% CH4 on total GP） were investigated by meta-analysis. These factors were considered：pressure in the GP equipment（0 = constant; 1 = increasing）, incubation time（0 = 24; 1 = ≥ 48 h）, time of rumen fluid collection（0 = before feeding; 1 = after feeding of donor animals）, donor species of rumen fluid（0 = sheep; 1 =bovine）, presence of N in the buffer solution（0 = presence; 1 = absence）, and ratio between amount of buffered rumen fluid and feed sample（BRF/FS; 0 = ≤ 130 mL/g DM; 1 = 130–140 mL/g DM; 2 = ≥ 140 mL/g DM）. The NDF content of feed sample incubated（NDF） was considered as a continuous variable. From an initial database of 105 papers, 58 were discarded because one of the above-mentioned factors was not stated. After discarding 17 papers,the final dataset comprised 30 papers（339 observations）. A preliminary mixed model analysis was carried out on experimental data considering the study as random factor. Variables adjusted for study effect were analyzed using a backward stepwise analysis including the above-mentioned variables. The analysis showed that the extension of incubation time and reduction of NDF increased GP and CH4 values. Values of GP and CH4 also increased when rumen fluid was collected after feeding compared to before feeding（＋26.4 and ＋9.0 mL/g DM, for GP and CH4）,from bovine compared to sheep（＋32.8 and ＋5.2 mL/g DM, for GP and CH4）, and when the buffer solution did not contain N（＋24.7 and ＋6.7 mL/g DM for GP and CH4）. The increase of BRF/FS ratio enhanced GP and CH4production（＋7.7 and ＋3.3 mL/g DM per each class of increase, respectively）. In vitro techniques for measuring GP and CH4 production are mostly used as screening methods, thus a full standardization of such techniques is not feasible. However, a greater harmonization of analytical procedures（i.e., a reduction in the number of available protocols） would be useful to facilitate comparison between results of different experiments.

Effects of Condensed Tannins from Quebracho Extract on the Kinetic of in vitro Gas Production on Trifofium repens, Lotus corniculatus and Lofium perenne

The aim of this work was to study the effect of condensed tannins （CT） on the kinetic of gas production in vitro, on the three main species of Azorean pasture： Trifolium repens, Lotus corniculatus and Lolium perenne, using Quebracho extract as the source of CT, and also to calculate total tannins and condensed tannins on the species studied. Lotus corniculatus, Lolium perenne and Trifolium repens were tested in vitro with the inclusion of Quebracho extract in doses of 0%, 2.5% and 5% DM. The total content in tannins was determined in the 3 species. We have found, in comparison with standard tannic acid, that tannins exist in the flower of T. repens （0.81 equivalents of tannic acid） and in L .corniculatus （1.07 equivalents of tannic acid） but not in L .perenne （0 equivalents of tannic acid）. The concentration of condensed tannins in the 3 species was： 0.34 mg/mL for T. repens; 0.83 mg/mL for L. corniculatus and 0 mg/ml for L. perenne. We verified that a significant reduction （P 〈 0.05） of gas production happened for doses of 5% of CT in comparison with the other two concentrations： 0% and 2.5% of CT. The results of this study express a reduction of the fermentation rate, which implies a reduction of gas production, so, a reduction of methane emission to the atmosphere and an increase of exploitation of the protein by ruminants.

Controlled Freezing and Open-Pulled Straw (OPS) Vitrification of In vitro Produced Bovine Blastocysts FollowingAnalysis of ATP Content and Reactive Oxygen Species (ROS) Level

To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.

Supplementation of L-ascorbic acid improves the in vitro development of buffalo(Bubalus bubalis)embryos and alters the expression of apoptosis-related genes

Objective:To study the effect of L-ascorbic acid supplementation on the in vitro development of buffalo embryos and evaluate the relative mRNA abundance of some pro-apoptotic,anti-apoptotic,and embryonic development-related genes.Methods:In experiment 1,we evaluated the effect of the addition of 0(control),50,and 100μM L-ascorbic acid to the in vitro maturation medium on the developmental competence in terms of blastocyst rate and relative mRNA abundance of some pro-apoptotic(BAX,BID),anti-apoptotic(BCL-XL,MCL1),and embryonic development(GDF9,BMP15)related genes.Based on the results,we chose 50μM as the suitable dose of L-ascorbic acid for the subsequent experiments.We further evaluated the blastocyst rates following the addition of 50μM L-ascorbic acid to the in vitro culture medium(experiment 2),and in vitro maturation and in vitro culture media(experiment 3).In all three experiments,the maturation and culture media devoid of L-ascorbic acid served as the control group.Results:The blastocyst rate after adding 50μM L-ascorbic acid to the in vitro maturation medium was significantly higher than the control group(P<0.05),whereas 100μM L-ascorbic acid exhibited a negative effect on the blastocyst rate.The blastocyst rates for embryos cultured in 50μM L-ascorbic acid in the in vitro culture medium alone and both in vitro maturation and in vitro culture media were significantly higher than their corresponding control groups(P<0.05).The relative mRNA abundance of BAX significantly decreased in blastocysts produced after the addition of 50μM L-ascorbic acid as compared with the control group(P<0.05),whereas,for MCL1,it significantly decreased in blastocysts produced after the addition of 100μM L-ascorbic acid(P<0.05).Conclusions:The supplementation of 50μM L-ascorbic acid to in vitro maturation and in vitro culture media supports in vitro embryonic development in buffaloes by improving developmental competence and altering the expression of apoptosis-related genes.

Overload type and optimization of meadow carrying capacity in Maqin County in the Three-River Source Region,China

Alpine meadows,comprising the main ecosystem of the Three-River Source Region,play an important role in the economic development of western pastoral areas.To determine whether the grazing level was too intense or the nutrient-carrying capacity of the meadows was overloaded,and to offer solutions in Maqin County,Qinghai Province,China,the meadows carrying capacity was determined by combining grass-yield and nutrients data for different seasons across different meadows.The results showed that the levels of crude protein(CP),ether extract(EE),ash,soluble sugars(SS),gas production,and energy value of forage in the summer–autumn grazing meadows were higher than those of the winter–spring meadows.Neutral detergent fiber(NDF)and acid detergent fiber(ADF)of forage in the summer–autumn grazing meadows were lower than those of the winter–spring meadows.We found that no pastures were overloaded with metabolizable energy(ME)for the whole year but digestible crude protein(DCP)was overloaded in winter–spring.And there were differences in the amount carrying capacity(forage yield)of seasonal grazing meadows for the different herds,the overload types were annual overloading(herd A),overloaded in summer–autumn(herd B),overloaded in winter–spring(herd C),and not overloaded(herd D).Compared with the previous grazing mode,theoretical carrying capacity,DCP carrying capacity,and ME carrying capacity of herd A increased by 20,19,and 27 sheep units,respectively,after optimization;herd B:36,31,and 45 sheep units,respectively;herd C:28,23,and 44 sheep units,respectively;and herd D:43,40,and 61 sheep units,respectively.In the Three-River Source Region and similar alpine pastoral areas,the grassland-livestock structure should be optimized to improve grassland vegetation status and increase the theoretical carrying capacity.

Effect of toasting grain silages from field peas(Pisum sativum)and field beans(Vicia faba)on in vitro gas production,methane production,and post-ruminal crude protein content

Legume grains such as field peas and field beans can be produced on a local level,and may be reliable sources of dietary protein and energy apart from common soybean and rapeseed meals.In ruminants,protein,starch,and carbohydrates from peas and field beans are fermented in large part before reaching the small intestine.The objective of this study was to evaluate the effects of a combination of ensiling and hydro-thermic treatment(i.e.,toasting at 160℃for 30 min)of grains of peas and field beans on the concentrations of post-ruminal crude protein(PRCP)and rumen-undegraded protein(RUP).Moreover,24-h gas production and methane production were measured.For this,an in vitro batch culture system with ruminal fluid from sheep was used.Rumen-undegraded protein was determined using the Streptomyces griseus protease test.Scanning electron micrographs were used to visualize morphological changes of starch granules and their joint matrices in peas and field beans after ensiling,toasting,or a combination of both.Native pea grains contained crude protein(CP)at 199 g/kg DM,PRCP at 155 g/kg DM at a ruminal passage rate of 0.08/h(Kp8),RUP at 33 g/kg DM at Kp8,and starch at 530 g/kg DM.Native field beans contained CP at 296 g/kg DM,PRCP at 212 g/kg DM at Kp8,RUP at 54 g of/kg DM at Kp8,and starch at 450 g/kg DM.The PRCP did not considerably differ among native and treated peas or field beans.Especially in the peas,RUP at Kp8 increased after ensiling by 10 g/kg DM(i.e.,30%;P<0.05).Toasting increased RUP(Kp8)in ensiled peas by another 28%(P<0.05).Toasting had no effect on PRCP or RUP when the peas or field beans were not ensiled before.Gas and methane production were not affected by any treatment,and scanning electron micrographs did not reveal structural changes on the starches doubtless of any treatment.Protein seemed to be more affected by treatment with ensiled+toasted peas than with ensiled+toasted field beans,but starches and other carbohydrates from both legumes remained unaffected.

Rumen liquor from slaughtered cattle as inoculum for feed evaluation

Use of nonlinear mathematical models has been majorly based on in vitro gas production(GP) data generated when substrates are incubated with rumen liquor from fistulated steers. However,existing evidence suggests that rumen liquor from slaughtered cattle of unknown dietary history also generates quantifiable in vitro GP data. Fitting and description of GP data obtained from 4 diets incubated with rumen liquor from slaughtered cattle was evaluated using single-pool exponential model with discrete lag time(EXPL), logistic(LOG), Groot's(GRTS) and Gompertz(GOMP) models. Diets were formulated by varying proportions of Rhodes grass(Chloris gayana) hay and a concentrate mixed on dry matter basis to be: 1,000 g/kg Rhodes grass hay(RGH) and 0 of the concentrate(D1), 900 g/kg RGH and 100 g/kg concentrate(D2), 800 g/kg RGH and 200 g/kg concentrate(D3), 700 g/kg RGH and 300 g/kg concentrate(D4). Dietary kinetics for the models were determined by measuring GP at 2,4.8,10.18,24.36,48.72,96 and 120 h. Model comparison was based on derived GP kinetics, graphical analysis of observed versus predicted GP profiles plus residual distribution and goodness-of-fit from analysis of root mean square error(RMSE), adjusted coefficient of determination(Adj-R^2) and Akaike's information criterion(AIC). Asymptotic GP, half-life and fractional rate of GP differed(P < 0.001) among the 4 models. The RMSE, Adj-R^2 and AIC ranged from 1.555 to 4.429,0.906 to0.984 and 2.452 to 15.874, respectively, forall diets compared across the 4 models. Based on the goodness-of-fit statistical criterion, GP profiles of D1 were more appropriately fitted and described by GRTS and GOMP than the EXPLand LOG models. The GRTS model had the lowest AIC value for D2(2.452). Although GRTS model had the most homogenous residual dispersion for the 4 diets, all the 4 models exhibited a sigmoidal behavior.Therefore, rumen liquor from slaughtered cattle of unknown dietary history can be used to derive nutritionally important feed parameters, but choice of the most appropriate model should be made based on fitting criteria and dietary substrates incubated.