Effects Of Sperm Pretreatment And Maternal Heat Stress On Developmental Competence Of Bovine Uniparental Embryos

In vitro production of the embryos, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), somatic nuclear transfer and uniparental (parthenogenetic and androgenetic) embryos, is quite important for maximizing the utilization of animal reproductive performance, accelerating the genetic improvement, producing genetic modified animals and studying the developmental events involved in the early embryonic development. The uniparental embryos, containning genetic materials only from one of the parents, are widely used for studying genomic imprinting, establishing uniparental embryonic stem cells and producing genetic modified animals in murine. Recently, androgenetic embryos in livestock were coming out on stage, however, the efficiency to produce was extremely low. The defect of sperm decondensation within the ooplasm was proposed as one of the key factors disrupted development of androgenetic embryos in livestock. In normal fertilization, sperm always undergo some important physiological processes, such as capacitation, acrosome reaction and the fusion of plasma membrane of gametes. To mimic those events, capacitating sperm and removing acrosome and plasma membrane prior to sperm injection may thus enhance decondensation protential of injected sperm and developmental competence of androgenetic embryosElevated temperature in summer can disrupt oocytes development in female cattle, and lead to the reduction in reproductive performance and developmental competence of IVF embryos produced with the oocytes. Thus, maternal heat stress may have some impacts on uniparental embryos as well. Besides, the season, which was frequently used to describe the environment that female cattle exposed to in previous studies, may not be an accurate indicator for quantifying the magnitude of heat stress. Temperature-humidity index (THI), as an indicator for weighing the magnitude of heat stress, was widely used in determining the effects of heat stress on milk yield and conception rates of dairy cattle. Thus, the results could be more comparable and referenceable if use THI to evaluate effects of heat stress on the quality and subsequence development of oocytes. It has been reported that betaine can help microbes, plants and poultry to alleviate heat stress, its roles in mammals remains unkown. Identifying physiological functions of betaine with an in vitro cultured cells model could lay a foundation for its protential use in enhancing thermotolerance of in vitro produced bovine embryos.1. Effects of sperm pretreatments on the status of acrosome and plasma membraneThe objective of this study was screening for a efficient and save method to remove acrosome and plasma membrane of sperm, piolot analysis of effects of those pretreatments including capacitation on sperm decondensation capacity. The acrosome and plasma membrane of bovine sperm were labeled with lectins (FITC-PNA and FITC-WGA, respectively). Effects of capacitation and treatment of calcium ionophore A23187(Ca A23187), Triton X-100(TX-100) and lysolecithin (LL) on status of acrosome and plasma membrane were quantitatively analyzed. Effect of removal of acrosome and plasma membrane on sperm viability and integrity of chromatin was evaluated with the live/dead assay and acridine orange staining. In an oocyte-free system, effect of sperm capacitation and/or membranes-removing treatment on sperm decondensation protential was determined. Main results are shown as follows:(1) The acrosome-removed rates of TX-100-(61.53±2.06%) and LL-(51.87±2.53%) treated sperm were significantly higher (P<0.05) comparing to control(25.81±2.99%) and Ca A23187-treated sperm (33.10±4.73%). The plasma-membrane-removed rates of LL-treated sperm was the highest (P<0.05) comparing to other three groups. Sperm capacitation significantly (P<0.05) efficiency of removing acrosome and plasma membrane of sperm.(2) The removal of acrosome and plasma membrane led to a reduction of sperm viability, but did not damaged sperm chromatin.(3) The porprotion of sperm, which were not senstive to the decondensation induction of DTT, was significantly lowered by TX-100and LL treatment comparing to control (6.11±1.00%vs.20.30±0.93%, P<0.05;3.51±0.82%vs.20.30±0.93%, P<0.05). Combined capacitation with LL could significantly increase the porprotion of sperm which entered an advanced decondensation stage (54.60±1.98%vs.15.62±1.61%, P<0.05).2. Effects of sperm capacitation combined with removal of acrosome and plasma membrane on the development of bovine androgenetic embryosThe objective of this study was further analysis of effects of capacitation and removal of acrosome and plasma membrane prior to sperm injection on decondensation protential, synchronism of sperm chromatin remodling and embryonic development of sperm clonging derived androgenetic (SpC-AG) embryos. The embryos were grouped with the pretreatments of injected sperm, which were control, capacitation (CM), TX-100, LL, CM+TX-100and CM+LL. At first, the decondensation stages of injected sperm within the embryo were observed using Hoechst33342staining. Then, development of SpC-AG embryos was recorded daily. To investigate developmental synchronism between nuclear and ooplasm, nucleus of cleaved embryos were visualized with fluorescent staining. At last, the morulas and blastocysts were transferred into recipients. Main results are shown as follows:(1) Synchronized decondensation of the two injected sperm within SpC-AG embryos was significantly improved in LL group comparing to control (51.1%vs.23.3%. P<0.05). Combination of capacitation and LL showed the highest synchronized decondensation rates (72.2%) and pronuclear formation rates (46.3%) of SpC-AG embryos.(2) It is the first report of producing bovine SpC-AG embryos at blastocyst stage. Injection of sperm treated with TX-100or LL significantly (P<0.05) reduced ooplasmic fragmentation rates of SpC-AG embryos. LL group embryos showed higher(P<0.05) rates in develop to8-cell stage, morula stage and blastocyst stage comparing to control. Injection with capacitated and capacitated combined with TX-100-or LL-treated sperm could increase (P<0.05) blastocyst development of SpC-AG embryos.(3) LL treatment prior to sperm injection enhanced sychronism of nuclear-ooplasimc development within the cleaved SpC-AG embryos comparing to control (64.1%vs.19.7%, P<0.001). At the mean time, unchanged sperm head was less detected in LL group (10.9%vs.47.5%, P<0.001).(4) All transferred SpC-AG embryos fail to establish pregnancy in recipients. However, return-to-estrus of recipients was delayed, indicating embryo loss at the early stage of pregnancy.3. Effects of maternal heat stress on the development of bovine uniparental embryosTo evaluate effects of maternal heat stress on development of bovine uniparental embryos, in this study, THI instead of season was used as the indicator of environmental heat stress. The magnitude of heat stress was grouped as no stress (THI<72), mild stress (72≤THI<80) and moderate-severe stress (THI≥80). The effect of heat stress of oocyte donors on in vitro maturation (IVM) rates of2585oocytes and subsequent developmental capacity of2807ICSI, parthenogenetic and androgenetic embryos. The correlation between1) THI and oocyte IVM rates and2) THI and embryonic development rates were analyzed. Main results are shown as follows:(1) The environmental THI during follicular development had impact on IVM rates of oocytes (P<0.05), and a negative correlation was evident between THI and IVM rates (r=-0.37). IVM rates did not affected by mild heat stress (72<THI<80), but did by moderate-severe stress (THI>80;52.1±2.9%vs63.2±2.3%, P<0.01, comparing to THI<72).(2) The environmental THI during follicular development showed impact on cleavage rate (P<0.01),4-8cell rates (P<0.001) and>16cell rates (P<0.001) of ICSI embryos. THI was found negative correlated with4-8cell rates (r=-0.52) and>16cell rates (r=-0.40). Moderate-severe heat stress (THI>80) significantly decreased (P<0.05) development of ICSI embryos at all stages.(3) The environmental THI during follicular development showed impact on4-8cell rates (P<0.01) and>16cell rates (P<0.05) of parthenogenetic embryos, but not on cleavage rates. THI was found negative correlated with4-8cell rates (r=-0.41) and>16cell rates (r=-0.45). Under heat stress (THI>72),4-8cell rates of parthenogenetic embryos were significant reduced (P<0.05) comparing to free of heat stress (THI<72). Under moderate-severe heat stress (THI>80), both4-8cell rates and>16cell rates were significantly declined (P<0.05).(4) The environmental THI during follicular development showed no effect on development of androgenetic embryos.4. The physiological function of betaine during allevation of heat stressThe objective was to examine whether betaine played a role in three-dimensional cultured bovine mammary epithelial cells (BMECs) during heat stress. Effects of betaine treatment on the heat stres responses, apoptosis, proliferation and viability of BMECs under thermoneutral (TN) and heat stress (HS) conditions was investigated using real-time PCR, TUNEL and XTT assay, respectively. This study provided a physiological function of betaine which could be protentially used in helping bovine embryos and even animals to survive from elevated temperature. Main results are shown as follows:(1) In HS, treating with betaine, morphology of BMECs was maintained, apoptosis rates were significantly decreased (P<0.0001), and viability was increased (P<0.05) in contrast. (2) In HS, the production ability of HSP70and HSP27in BMECs was maintained with betaine treatment.(3) In TN, the requirement of HSPs in BMECs was reduced with betaine treatment, and therefore, cell proliferation rates were accelerated.In conclusion, the combined pretreatment of capacitation and lysolecithin efficiently removed acrosome and plasma membrane of bovine sperm, and thus enhanced sperm decondensation capacity within the injected oocytes and improved developmental competence of bovine androgenetic embryos; The environmental THI during follicular development of donor cattle was capable for indicating maternal heat stress, developmental competence of ICSI and parthenogenetic embryos was decreased by maternal heat stress; The nagetive impacts of heat stress, including apoptosis, reduced viability and HSP expression capacity, were alleviated with betaine treatment in BMECs, indicating a protential use of betaine for counteracting heat stress in dairy cattle.