(PhD in Animal Nutrition & Physiology) By: A. Riasi (PhD in Animal Nutrition & Physiology) http://riasi.iut.ac.ir Isfahan University of Technology, Isfahan, Iran Advanced Reproductive Physiology (part 4)

Spermatozoa in female tract In natural mating semen are introduced in: Vagina Cervix Within the female tract spermatozoa are lost by: Neutrophils’ phagocytosis Physical barrier including the cervix

Spermatozoa in female tract Two stages for spermatozoa transportation: Rapid Effect of oxytocin secretion Effect of prostaglandins Sustained

Spermatozoa in female tract Factors may affect spermatozoa transport in cervix: Sperm motility Physicochemical change in cervix secretions Sulfomucin Sialomucin

Spermatozoa in female tract Sperm capacitation: Chemical changes Remove decapacitation factors Remove cholesterol Membrane ions changes Physical and morphological changes

Spermatozoa in female tract Higher levels of fertilization promoting peptide (FPP) prevent capacitation FPP is found in the seminal fluid. FPP comes into contact with the spermatozoa upon ejaculation. FPP has a stimulatory effect on adenosine which increases adenylyl cyclase activity in the sperm.

Spermatozoa in female tract Other chemical changes: Removal of cholestrol and non-covalently bound epididymal/seminal glycoproteins is important. Increased permeability of sperm to: Ca2+, HCO3− and K+ The increase of intracellular cAMP levels.

Spermatozoa in female tract Altering the lipid composition of sperm plasma membranes affects: The ability of sperm to capacitate Acrosomal reaction Respond to cryopreservation

The oocyte moving in female tract Oocyte is transported by cilia of oviduct. Smooth muscles of oviduct adjust the time of oocyte transportation. The mature egg can only survive for about 6 hours, so the time of insemination is important.

Sperm penetration A series of events take place in AIJ: Acrosomal reaction The vesicles sloughed, leaving the inner acrosomal membrane and the equatorial segment intact

Sperm penetration A spermatozoon has to penetrate four layers before it fertilizes the oocyte:

Sperm penetration

Sperm penetration Two different proteins in sperm membrane: ZBR ARPR Reaction ZBR and ZP3 facilitate the sperm and zona pellucida contact. ARPR Reaction of ARPR and ZP3 start the acrosomal reaction

Sperm penetration

Sperm penetration

Sperm penetration Three changes occur in the oocyte after penetration of vitelline membrane:

Fertilization Fertilization has two important genetic consequences: The diploid chromosome number is restored (2n). The genetic sex of the zygote is determined

Cleavage

Cleavage Blastomeres formation Totipotency potential

Cleavage Dissociation and reaggregation of morula-stage blastomeres. (A) There is clear evidence for molecular differences between cells (ie, specification) as early as the four- to eight-cell stage [61–71,157,158]; yet, cell fates remain plastic. (B, C) Following dissociation, it is likely that a combination of cell sorting (arrows) and respecification restores the original status of the embryo. (D) Once cell–cell contacts have been reformed, development proceeds from the blastocyst stage. (E) If only ‘‘inner’’ or ‘‘outer’’ (shown) cells are reaggregated, cell sorting (arrows) is still likely to occur. (F) Outer cells remain competent to produce both ICM cells and additional TE cells (curved arrows), as they do in normal development. (G) A combination of cell sorting, regeneration of ICM cells from outer cells, and (potentially) respecification of outer cells to an ICM lineage restores the original status of the embryo. (Adapted from Maureen L. Condic, Stem Cells and Development , 2014 )

Cleavage

Increase conception rate Embryonic mortality in the initial seven days of gestation: Fertilization failure Genetic defects Impaired embryonic development

Increase conception rate Measuring embryonic mortality in weeks two and three of gestation is much more challenging. This period coincides with the maternal recognition of pregnancy.

Increase conception rate Successful establishment of pregnancy depends on a delicate balance between: Luteolytic mechanisms inherent to the endometrium at the end of diestrus. Antiluteolytic mechanisms, orchestrated by the conceptus.

Increase conception rate Some strategies for increasing conception rate: Using TAI protocols Stimulate growth and/or differentiation of the pre-ovulatory follicle Stimulate CL growth rate Increase plasma progesterone concentrations in the initial three weeks after insemination.

Increase conception rate Decrease the effects of a dominant follicle during the critical period Antiluteolytic stimulus provided by the conceptus Decrease uterine luteolytic capacity

Increase conception rate Some factors may affect Ovsynch results: The stage of the estrous cycle Cyclic status at the time that GnRH is administered (Bisinotto et al., 2010)

Increase conception rate Researchers have modifed the original Ovsynch protocol to try to: Improve synchrony and fertility through presynchronization Altering the timing of AI in relation to ovulation Testing the various injection intervals of the original protocol

Increase conception rate TAI programs need day-to-day operation, so it may use for: Lactating dairy cows with little or no estrus detection at all Voluntary Waiting Period (VWP)

Increase conception rate Factors explaining the variation in conception rate to TAI among herds may include: The proportion of anovular cows The follicular dynamics of individual cows The ability of farm personnel to implement Ovsynch

Increase conception rate Following this first report, numerous protocols have been proposed and routinely applied in high production dairy cows (Wiltbank et al., 2011).

Increase conception rate Programming cows for first postpartum AI using presynch/ovsynch Use of presynch for programming lactating dairy cows to receive their first postpartum TAI can improve first service conception rate in a dairy herd.

Increase conception rate One possible hormone injection and TAI schedule for the Presynch/Ovsynch protocol based on the results of Moreira et al., 2000

Increase conception rate In an assay, cycling cows conception rate was 29% for Ovsynch and 43% for Presynch. These protocols may presents low efficiency when applied in tropical condition.

Increase conception rate Estradiol plus progesterone based protocol Exogenous P4 (progestins and Melangestrol acetate) has consequences: Suppresses LH release Alters ovarian function Suppresses estrus Prevents ovulation

Increase conception rate Several studies found that P4 treatment suppress the growing phase of the dominant follicle. In E2 plus P4 protocols, a lower dose of E2 is normally given from 0 to 24 h after progestin removal to induce a synchronous LH surge.

Increase conception rate Anestrous cows have insufficient pulsatile release of LH to support the final stages of ovarian follicular development and ovulation. What we should do for TAI in anestrous cows? The treatment with equine chorionic gonadotropin (eCG) may be effective.

Increase conception rate Antiluteolytic strategies after AI: Pharmacological Mechanical Nutritional Management

Increase conception rate Strategies to increase progesterone after AI: Daily injection of progesterone Using of progesterone releasing intravaginal device (PRID) during the days 5-9 after AI. Inducing the formation of accessory corpora lutea using GnRH, LH or hCG and for ovulation of the first wave dominant follicle.

Increase conception rate Effect of estrogen Estrogen secretion from a large follicle during the days 14-17 after AI may affect embryo survival. This hormone has a central role in PGF production and luteolysis.

Increase conception rate Some strategies for reducing estrogen after AI: Absence of dominant follicles Reduction of their steroidogenic capacity Reduction of endometrial responsiveness to estradiol during the period of maternal recognition of pregnancy Pharmacological approaches

Increase conception rate Pharmacological strategies The GnRH-hCG treatment It induced an increase in plasma progesterone concentrations

Increase conception rate Antiluteolytic strategies: Antiinflamatory drugs Fat feeding Bovine somatotropin (bST)

Increase conception rate Synthesis of PGF results from a coordinated cascade of intracellular events. A rate limiting step in this cascade is the conversion of arachidonic acid to prostaglandin-H2 (PGH).

Increase conception rate

Increase conception rate

Increase conception rate Fat feeding influences several aspects of reproduction in cattle (See review by Santos et al., 2008).

Increase conception rate Feeding long chain fatty acids can modulate PGF production in the endometrium. Effect of n-3 fatty acids (Mattos et al., 2003, 2004) Effect of n-6 fatty acids (Pettit and Twagiramungu, 2004) A summary of the effects of fatty acid feeding on cattle fertility reported by Santos et al. (2008).

Increase conception rate Strategies for growth of the conceptus Secretion of IFN is positively associated with conceptus size. Administration of bST.

Maternal recognition of pregnancy Mother quickly becomes cognizant of the cleavage-stage embryo within her body. Mother reacts to embryo presence, but its not enough for the pregnancy to proceed.

Maternal recognition of pregnancy For maternal recognition it is necessary: The normal cyclic regression of CL be prevented in order to maintain progesterone production. The conceptus has also to ensure that an adequate supply of maternal blood reaches the sites of placentation.

Maternal recognition of pregnancy The conceptus is recognized as foreign by the mother and it must nevertheless take steps to avoid a losing confrontation with the maternal immune system. The conceptus does not become vascularized by the host's blood supply.

Maternal recognition of pregnancy In horses The equine conceptus forms an encapsulated spherical structure between days 12 and 14. The constant patrolling may be the key to the mechanism that inhibits PGF2α release. Maternal recognition of pregnancy

Maternal recognition of pregnancy In cattle and sheep The conceptus begins to intervene in the luteolytic process three to four days before the CL actually become dysfunctional. In these species, the antiluteolytic substance, an unusual Type I interferon (IFN)-t, has been reviewed on numerous occasions in the literature. Its presence in the lumen clearly suppresses the normal pattern of pulsatile release of PGF2α. Maternal recognition of pregnancy

Maternal recognition of pregnancy Importance of progesterone: The concentrations of progesterone at a critical time before implantation is important for cows pregnancy. Two logical possibilities for lower progesterone in the lactating dairy cows: Secretion by the corpus luteum is reduced Metabolism of progesterone is increase

Maternal recognition of pregnancy Importance of progesterone: Some factors may affect the metabolism and excretion of progesterone: Feed intake Milk yield Administration of exogenous progesterone

Embryonic loss Much prenatal mortality occurs in all mammals. Higher amount of embryonic wastage occurs following IVF and ET. The majority of these losses occur prior to or during implantation.

Embryonic loss Embryonic losses in sheep and cattle: It most occurring in the first 3 wk of pregnancy. Natural asynchronies: The late onset of the first meiotic division may lead to some oocytes being delayed in their maturation. A second natural cause of asynchrony may be due to delayed fertilization. Finally, embryos are known to cleave at different rates.

Embryonic loss Injection interferons have ability to improve pregnancy success in ewes may be due: The rescue of embryos delayed.

Pregnancy-Associated Glycoproteins (PAG) In 1982 the partial purification and characterization of a pregnancy-specific protein (PSP-B) was reported from cattle. More recently, isolated several isoforms of PAG from bovine placental tissue.

It is now clear that PSP-B and PAG-1 are identical in sequence. Pregnancy-Associated Glycoproteins (PAG) It is now clear that PSP-B and PAG-1 are identical in sequence. The presence of PAG-1 (or PSP-B) in blood serum has provided the basis of a potentially useful pregnancy test in cattle.

The antigen generally becomes detectable by about day 20 postbreeding. Pregnancy-Associated Glycoproteins (PAG) The antigen generally becomes detectable by about day 20 postbreeding. In cattle, concentrations of the antigen rise gradually during gestation and peak just prior to parturition.

The PAG have a well-defined peptide- binding cleft. Pregnancy-Associated Glycoproteins (PAG) The PAG have a well-defined peptide- binding cleft. They are relatively hydrophobic polypeptides. They are unlikely to have enzymatic activity.

Two possible functions for PAG are suggested: Pregnancy-Associated Glycoproteins (PAG) Two possible functions for PAG are suggested: They could be hormones, which, by virtue of their binding clefts, are able to bind specific cell surface receptors on maternal target cells. The second suggestion is that PAG sequestered or transported peptides

Some research papers associated to this lecture 1-Pancarci, et al. 2002. Use of estradiol cypionate in a presynchronized timed artificial insemination program for lactating dairy cattle. J. Dairy Sci. 85:122–131. 2- Franco, et al. 2006. Effectiveness of administration of gonadotropin-releasing hormone at Days 11, 14 or 15 after anticipated ovulation for increasing fertility of lactating dairy cows and non-lactating heifers. Theriogenology 66: 945–954. 3- De Rensis, et al. 2008. Inducing ovulation with hCG improves the fertility of dairy cows during the warm season. Theriogenology 69: 1077–1082 4- Bartolome, et al. 2005. Strategic use of gonadotrophin-releasing hormone (GnRH) to increase pregnancy rate and reduce pregnancy loss in lactating dairy cows subjected to synchronization of ovulation and timed insemination. Theriogenology 63: 1026–1037.