P. D. Burns

Effect of heparin on cleavage rates and embryo production with four bovine sperm preparation protocols

Heparin is often added to fertilization media to induce sperm capacitation. However, recent observations from the bovine IVF industry have indicated that heparin may not be necessary to induce sperm capacitation when cryopreserved bovine sperm are separated through Percoll gradients. The objective of these studies was to determine if the addition of heparin to fertilization media was required following separation of frozen-thawed bovine sperm. Experiment 1 was conducted to determine the effect of heparin on cleavage rates and embryo production when using Percoll, BSA or washing sperm separation protocols. Experiment 2 was conducted to determine the effect of heparin on cleavage rates and embryonic development when using a new sperm separation protocol (PureSperm). In Experiment 1, regardless of sperm separation protocol, heparin increased cleavage rates and embryo production (P<0.05). Further, cleavage rates and embryo production were higher for the Percoll procedure compared to either BSA or washing sperm separation protocols (P<0.05). In Experiment 2, there was no difference between Percoll and PureSperm separation protocols on cleavage rates or embryo production (P>0.05). However, heparin increased cleavage rates for both protocols and improved embryo production for the PureSperm protocol (P<0.05). In conclusion, use of heparin in fertilization media improves in vitro embryo production when using cryopreserved bovine sperm. Furthermore, PureSperm can be used as an alternative to Percoll for bovine sperm separation.

Cellular Mechanisms by Which Oxytocin Mediates Ovine Endometrial Prostaglandin F2α Synthesis: Role of Gi Proteins and Mitogen-Activated Protein Kinases

Abstract Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2α synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2α synthesis. The objective of experiment 1 was to determine whether Gi proteins mediate oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of Gi proteins, had no effect on the ability of oxytocin to induce PGF2α synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF2α synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF2α synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to Gi proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2α synthesis in ovine endometrium.

Cellular mechanisms by which oxytocin stimulates uterine PGF2α synthesis in bovine endometrium: Roles of phospholipases C and A2

The objective of these experiments was to identify the cellular mechanisms by which oxytocin stimulates prostaglandin (PG) F2 alpha synthesis in bovine endometrial tissue. Uteri were collected on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to assess PGF2 alpha release or phospholipase (PL) C activity. Oxytocin (10(-6) M) stimulated PGF2 alpha release and PLC activity within 30 min of incubation (P < 0.01). The highest stimulation was observed at 100 min (P < 0.01). Oxytocin stimulated PLC activity at 10(-9) M and higher doses, whereas an increase in PGF2 alpha release was not detected until 10(-8) M (P < 0.09). Melittin, a stimulator of PLA2 activity, stimulated PGF2 alpha release at 10(-6) M and higher doses (P < 0.01). Aristolochic acid, an inhibitor of PLA2 activity, blocked the ability of oxytocin to stimulate PGF2 alpha release at 10(-5) M and higher doses (P < 0.01). Aristolochic acid (10(-4) M) reduced the stimulation of PGF2 alpha release induced by A1F4-, a nonspecific stimulator of G protein (10(-5) M) and melittin (10(-4) M; P < 0.05). Aristolochic acid had no effect on the ability of oxytocin or A1F4- to stimulate PLC activity (P > 0.10). By comparing the time course of stimulation and dose-response relationships between PGF2 alpha and PLC activity, it appears that oxytocin may stimulate PGF2 alpha secretion by activating PLC. The effects of melittin and aristolochic acid indicate that PLA2 may play a role in mediating the stimulatory effect of oxytocin on PGF2 alpha secretion, as well.

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2α synthesis in bovine endometrium: role of calcium

The objective of these experiments was to determine the role of Ca2+ during oxytocin-stimulated prostaglandin (PG) F2 alpha release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF2 alpha release. A23,187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF 2 alpha in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca2+ in the cytoplasm by inhibiting endoplasmic reticulum Ca2+/ATPase pumps) stimulated release of PGF2 alpha in a concentration-dependent manner as well (P < 0.13). Oxytocin (10(-6) M), AIF4- (a nonspecific activator of G-proteins; 10(-5) M), A23,187 (10(-5) M), and melittin (a stimulator of phospholipase A2; 10(-4) M) stimulated PGF2 alpha release when explants were incubated in Ca(2+)-free medium (P < 0.10); however, oxytocin, A23,187, or melittin were unable to stimulate PGF2 alpha release when explants were incubated in Ca(2+)-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AIF4- from stimulating phospholipase C activity (P < 0.08). CoCl2 (a nonspecific Ca2+ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca2+ channel blocker) prevented oxytocin from stimulating PGF2 alpha release (P < 0.05). Our results suggest that both extracellular and intracellular Ca2+ may be required for oxytocin to stimulate PGF2 alpha secretion in bovine endometrial tissue.

Effect of oxytocin on expression of cytosolic phospholipase A2 mRNA and protein in ovine endometrial tissue in vivo

The induction of endometrial prostaglandin (PG) F2alpha synthesis by oxytocin is dependent upon activation of phospholipase (PL) A2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF2alpha synthesis by inducing synthesis of cytosolic PLA2 (cPLA2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P < 0.01). However, endometrial concentrations of cPLA2 mRNA did not change following oxytocin administration (P > 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P < 0.01). Endometrial concentrations of cPLA2 protein were greater in the cytosolic than in the microsomal fraction (P < 0.01). Oxytocin did not affect the amount of cPLA2 protein in either fraction (P > 0.10). In conclusion, oxytocin did not effect expression of either cPLA2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF2alpha synthesis by activating cPLA2 protein that is already present in an inactive form.

Periovulatory Changes in Ovarian Metalloproteinases and Tissue Inhibitors of Metalloproteinases (TIMPS) Following Indomethacin Treatment

The periovulatory luteinizing hormone (LH) surge sets in motion a series of biochemical and biophysical changes culminating in follicular rupture and release of the oocyte. Among these biochemical events is a stimulation of prostaglandin production in conjunction with a stimulation of proteolytic enzyme activity (reviewed in Refs. 1, 2). The LH-stimulated increase in prostaglandins (PGs) has been suggested to be of pivotal importance in the ovulatory process (1, 2). Such a postulate is supported by reports that PGF2α (3, 4) and prostacyclin (5) are able to induce follicular rupture in the absence of gonadotropins. Further evidence for the role of PGs in ovulation is the observation that blockade of PG production by various PG synthase inhibitors (e. g., indomethacin) results in an inhibition of ovulation in the rat, rabbit, sheep, pig, primate, and human (reviewed in Refs. 1, 2). The mechanism(s) by which PGs may regulate follicular rupture, however, is unknown. It is hypothesized that local PG production may impact ovarian blood flow, smooth muscle contractility, oxygen-free radical formation, and action, as well as proteolysis associated with apical connective tissue degradation and oocyte extrusion (1, 2).

Effects of Human Chorionic Gonadotrophin Administration on Artificial Insemination Pregnancy Rates in Beef Heifers

Abstract The objectives of this study were to determine whether administering human chorionic gonadotropin (hCG) at 5 d post-AI improves fertility in beef heifers by increasing serum progesterone concentrations. Nulliparous crossbred beef heifers from separate breeding seasons [early (EBH; n = 48) and late (LBH; n = 48) breeding heifers] were stratified by BW, body condition score (BCS), and age within breeding season and randomly allotted to one of two treatments. All heifers were synchronized for standing estrus with the 7-11 MGA®-Select Synch protocol. Control heifers received a saline injection (3 mL; i.m.) and heifers in the treatment (hCG) group received hCG (3300 IU; i.m.) at 5 d post-AI. Blood samples were collected on d −35 and −28 to determine estrous cycling rates and for 5 consecutive d (beginning 4 d post-AI) for serum progesterone analysis. No treatment × breeding season interaction occurred; therefore, EBH and LBH data were pooled to analyze for main effects. First-service conception rates, estrous cycling status, and estrous cycling response were similar (P>0.10) for control vs hCG heifers. Serum progesterone concentrations were similar (P>0.10) on d 4, 5, and 6 post-AI for control vs hCG heifers; however, heifers receiving hCG had greater (P

Water Vapor Transport in Snow A 2-D Simulation of Temperature Gradient Metamorphism

In dry snow packs experiencing a temperature gradient, heat and mass transport is responsible for the metamorphism of ice crystals. The work presented herein investigates the influence of geometry, density, and temperature on the coupled, simultaneous heat and mass transport in idealized, two dimensional ice lattice cells. Mass transfer rates, mass flux rates, concentration and temperature distributions, and effective diffusion coefficients and thermal conductivities are presented as functions of temperature, geometry, density, and time. The results of the analysis show clearly that both the mass and heat transport are strongly dependent upon the ice lattice geometry. The effective diffusion coefficient is enhanced relative to the diffusion coefficient for water vapor in air for all of the two dimensional geometries studied. In addition, the preferential growth of branch grains has been verified using both static and dynamic, computer generated images of the ice lattice cells undergoing temperature gradient metamorphism.

Effects of Copper and Zinc Source on Performance, Carcass Characteristics, and Lipid Metabolism in Finishing Steers123

An experiment was conducted to determine the effects of Cu and Zn source on performance, trace mineral status, lipid metabolism, and carcass quality of finishing steers. In the study, 195 steers were blocked by origin, stratified by BW, and sorted into 24 pens. Pens within blocks were then randomly assigned to treatments in a 2 × 2 factorial arrangement. Factors were 10 mg of Cu/kg of dietary DM from CuSO4 or 10 mg of Cu/kg of dietary DM from organic Cu and 90 mg of Zn/kg of dietary DM from ZnSO4 or 36 mg of Zn/kg of dietary DM from organic Zn with 54 mg of Zn/kg of DM from ZnSO4. Steers were fed a high concentrate finishing diet until they reached an approximate BW of 580 kg. Diets were fed once daily in the morning to allow ad libitum access to feed throughout the day. Daily feed offerings were recorded and feed refusal was measured every 28 d. Body weights were recorded for each steer and blood samples were collected from 3 steers per pen every 28 d. Three weeks prior to slaughter, subcutaneous adipose tissue biopsies were taken from 1 steer per pen. Postharvest longissimus dorsi muscle samples were collected and evaluated for fatty acid composition. There were no Cu or Zn main effects or Cu × Zn interactions for ADG, DMI, or feed efficiency. The effect of Cu or Zn source was similar across treatments for hot carcass weight, dressing percent, rib eye area, subcutaneous fat thickness, kidney, pelvic, and heart fat, and marbling score. There was a Zn effect for calculated yield grade. Steers receiving organic Zn had a lesser (P = 0.03) calculated yield grade than steers receiving inorganic Zn. Serum cholesterol, plasma Cu and Zn concentrations, fatty acid composition of longissimus muscle and subcutaneous fatty acid synthase activity were similar across treatments. Results from this study indicate that trace mineral source had little influence on performance, carcass characteristics, and lipid metabolism.

Effects of Fishmeal Supplementation on Fertility and Plasma Ω-3 Fatty Acid Profiles in Primiparous, Lactating Beef Cows

A 2-yr study was conducted using 82 lactating, 2-yr-old, primiparous crossbred beef cows (yr 1, n = 40; yr 2, n = 42) to study the effect of fishmeal supplementation on reproductive performance. Cows were fed a corn silage-based diet supplemented with fishmeal (5% DM) or corn gluten meal (8.7% DM), beginning 25 d prior to the start of and continuing through the 90-d breeding season. Cows were artificially bred with semen from bulls of proven fertility 12 h after being detected in estrus. Jugular blood samples were taken from all cows on d 3 or 4, 9 or 10, and 15 or 16 following first insemination and analyzed for serum progesterone. During yr 2 of the study, additional blood samples were collected from four cows within each treatment group immediately before supplementation began (d 0) and at 7-d intervals for the first 35 d of supplementation. Plasma samples were analyzed for Ω-3 fatty acids. Serum progesterone concentrations did not differ between treatment groups following the first insemination (P>0.76). However, first service conception rates approached significance (NS; P=0.12) and tended to be greater for cows supplemented with fishmeal when compared with cows supplemented with corn gluten meal (76% vs 62%, respectively). Overall pregnancy rates at the end of the breeding season did not differ between the two treatment groups (P>0.64). In yr 2, within 7 d of supplementation, plasma eicosapentaenoate (EPA) and docosahexaenoate (DHA) were greater for cows supplemented with fishmeal (P<0.001). In conclusion, these data indicate that fishmeal supplementation alters plasma EPA and DHA (yr 2) and may improve first service conception rates in primiparous beef cows.