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,1
* School of Veterinary Medicine, University of California–Davis, Tulare 93274
Department of Animal Sciences, University of Florida, Gainesville 32611
1 Corresponding author: jepsantos{at}ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: embryo • gossypol • heifer • reproduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The 2 most important cotton species grown in the United States are Upland (Gossypium hirsutum) and Pima (Gossypium barbadense). Pima cottonseed is more nutrient dense than Upland cottonseed, but it contains more total gossypol (TG) and free gossypol (FG) and, because it is delinted and usually fed in a cracked form to cattle, the availability of gossypol from Pima is greater than that from Upland cottonseed (Santos et al., 2005). Therefore, the greater nutritional value of processed Pima cottonseed in diets fed to cattle is compromised by its increased gossypol content and availability; thus, feeding recommendations might differ from those for Upland cottonseed.
One of the potential negative effects of feeding excess FG is a potential decline in the reproductive performance of dairy cows. Santos et al. (2003) demonstrated that as plasma gossypol concentrations increased, the risk for pregnancy in lactating dairy cows fed diets containing different types of cottonseed declined. Furthermore, cows fed the high-FG diet had a reduced rate of pregnancy per AI and increased pregnancy loss. These negative effects of gossypol on fertility were also observed in dairy cows receiving embryos from heifers fed gossypol (Galvão et al., 2006), which suggests that negative effects early in embryo development persist later during maintenance of pregnancy. Coscioni et al. (2003a) observed no effects of increasing FG intake from 0 to 40 mg/kg of BW on follicle development and luteal function in Holstein heifers; however, feeding gossypol to heifers increased plasma and follicular fluid gossypol concentrations, and heifers consuming 40 mg of FG/kg of BW produced more underdeveloped and degenerated embryos on d 7 after AI (Coscioni et al., 2003a). Depressed fertility caused by gossypol in female cattle might therefore arise from direct or indirect effects on the developing embryo. In fact, work with in vitro-derived embryos has demonstrated the negative effects of gossypol during early embryo development (Zirkle et al., 1988; Brocas et al., 1997; Herández-Cerón et al., 2005). Findings by Velasquez-Pereira et al. (1998, 2002) indicated long-term accumulation of gossypol in the reproductive tissues of dairy heifers, especially the corpus luteum (CL) and endometrium, possibly because of the lipophilic properties of the molecule, which might accumulate in endometrial phospholipids and lipoproteins, and the cholesterol present in the CL. Because responses in vivo and in vitro might differ, particularly for FG that can be detoxified in the rumen, it is important to determine whether the detrimental effects of gossypol observed with in vitro-produced embryos are also observed with embryos in vivo.
The objectives of this study were to determine the effects of different dietary FG intakes from cracked Pima cottonseed, similar to those commonly observed in lactating dairy cows fed rations containing cottonseed, on embryo development and viability in vivo and in vitro in superovulated Holstein heifers. The hypothesis tested was that gossypol affects early embryo development and viability in vivo and in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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.
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Superovulatory Treatment
The ovulation of heifers was synchronized with the Ovsynch protocol with a progesterone insert: d 0, 100 µg of GnRH i.m. (Cystorelin, 50 µg/mL of gonadorelin diacetate tetrahydrate, Merial Ltd., Athens, GA) and a controlled internal drug-releasing insert containing progesterone (CIDR, Eazi-Breed, Pfizer Animal Health, New York, NY); d 7, 25 mg of PGF2
i.m. (Lutalyse sterile solution, 5 mg/mL of dinoprost tromethamine, Pfizer Animal Health) and the CIDR removed; d 9, 100 µg of GnRH i.m. Ultrasound examination (Aloka SSD 500 V, Aloka Co. Ltd., Tokyo, Japan) of ovarian follicles and CL was performed every 24 h, starting at the PGF2 injection, until ovulation was determined. Five days after ovulation, a CIDR was inserted and an i.m. injection of 2 mg of estradiol benzoate (β-estradiol,3-benzoate, E-8515, Sigma, St. Louis, MO) was administered 24 h later. A total of 300 mg of FSH (Folltropin-V, Bioniche Animal Health, Pullman, WA) was used per heifer in 8 i.m. injections in decreasing doses starting at 4 d after the estradiol benzoate treatment (d 10 after ovulation). Two i.m. injections of PGF2 were administered on the seventh and eighth FSH treatments, and the CIDR was removed concomitantly with the last FSH injection. A final injection of 100 µg of GnRH was administered 36 h after CIDR removal to induce ovulation. Heifers were inseminated by the same person twice, 12 h apart, with the first AI 12 h after the GnRH treatment. Semen from 2 sires was randomly allocated across all treatments.
Uterine Flush for Embryo Recovery
The uterine horns of heifers were flushed with approximately 700 mL of flushing solution (Sterile Filtered Embryo Flushing Solution, PETS Inc., Canton, TX) in a standard nonsurgical procedure on d 5 after induction of ovulation with GnRH. Initially, the embryo-collection catheter was placed in one of the uterine horns 1 to 2 in. cranial to the bifurcation of the uterus, and 20 mL of flushing solution was infused. This first 20 mL was recovered, and the amount was measured and immediately frozen for later analysis of TG concentration. Afterward, each uterine horn was flushed with approximately 350 mL of embryo collection solution. Collected embryos-ova were placed in embryo-holding solution and then counted and morphologically evaluated with a stereomicroscope (Nikon SU Z800, Nikon, Melville, NY) for fertilization, and embryos were graded based on quality, according to the International Embryo Transfer Society: 1 = excellent and good; 2 = fair; 3 = poor; 4 = degenerate and dead (Stringfellow and Seidel, 1998).
Differential Staining of Cells
Viable early morulae, grades 1 to 3, from all dietary treatments were stained with 2 distinct fluorochromes approximately 2.5 to 3.5 h after collection. Each embryo was individually transferred into a 40-µL drop of fluorescein diacetate (FDA; Sigma F7378, Sigma Diagnostics, St. Louis, MO) and propidium iodide (PI; P-1304, Molecular Probes Inc., Eugene, OR) for an incubation of 10 min each (Jones and Senft, 1985). All embryos were washed after each respective stain and then mounted onto microscope slides. The concentrations of FDA and PI working solution were 10 and 5 µg/mL, respectively. One drop of a 50% glycerol solution was placed on a microscope slide before placing the embryo. An epifluorescent microscope was used to count the number of blastomeres stained differentially with FDA and PI. The outer diameter of the zona pellucida of embryos was accessed with a 4-mm linear reticle.
Assignment to Culture Treatments
Early morulae from all dietary treatments were washed with Ham’s F-10 medium in 3 consecutive 45-µL drops in a sterile petri dish. Morulae from each treatment were randomly assigned to be cultured in vitro for 96 h in 45-µL drops of medium containing either 0 (CM) or 10 µg/mL (GM) of gossypol acetic acid (GAA). The CM medium contained 0.1% ethanol, which was the same concentration used to dilute GAA to 10 µg/mL in GM.
Ham’s F-10 culturing medium was prepared 12 h before embryo collection. Medium was prepared under sterile conditions and filtered through a sterile, nonpyrogenic 0.2-µm syringe filter (190–2520, Fisher Scientific, Pittsburgh, PA) after fetal bovine serum (16140–63, Invitrogen, Carlsbad, CA) and antibiotic antimycotic (A5955, Sigma) aliquots were added. Control and GAA medium contained 10% fetal bovine serum, 1% antibiotic-antimycotic, and 0.1% ethanol (D2-82948, Gold Shield Chemical Company, Hayward, CA). The pH of a subsample of the CM and GM media was measured immediately after preparation and used to ensure a final pH between 7.1 and 7.4. Embryo culture was performed in gamma-irradiated 4-well vented dishes (PETS Inc.), and the drops were covered with mineral oil (DMPS-2x, Sigma).
Embryos were incubated at 39°C, in 5% CO2, and 84.8% relative humidity. Temperature and relative humidity in the incubator were monitored twice daily with a traceable digital hygrometer-thermometer. Embryos were evaluated every 12 h and kept covered at all times.
Blood Sample Collection and Analyses of Plasma and Uterine Flush
Blood samples (20 mL) were collected from all heifers 24 h before the day of flushing by puncture of the coccygeal vein or artery with heparinized Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). Blood tubes were immediately placed in ice and transported to the laboratory within 1 h of collection. Tubes were centrifuged at 2,000 x g for 10 min at 5°C for plasma separation. The plasma was frozen at –25°C for later analyses.
The plasma and uterine flush solution were analyzed for TG and gossypol isomers by HPLC according to a technique described in Mena et al. (2004). Plasma samples were also assayed for concentrations of urea N (Urea Nitrogen Procedure No. 640, Sigma Diagnostics), glucose by direct measurement with a YSI Model 2700 Select Biochemistry Analyzer (Yellow Springs Instrument Co. Inc., Yellow Springs, OH), NEFA (Johnson and Peters, 1993) with a commercial kit (NEFA C, Wako Chemicals USA Inc., Richmond, VA), and BHBA with a commercial kit (Randox Laboratories Ltd., Antrim, UK).
Experimental Design and Statistical Analysis
The experimental design was a randomized incomplete block design. Heifers were blocked according to age and BW, and within each block were randomly assigned to 1 of the 3 treatments. For the analyses of embryo development and quality at collection, the donor heifer was considered the experimental unit of analysis. For the analyses of embryo staining immediately after collection, the embryo was used as the experimental unit of analysis. During in vitro culture of embryos, a 3 x 2 factorial arrangement of treatments was used, with 3 heifer treatments and 2 embryo culture medium treatments, and embryo was considered the experimental unit of analysis and donor heifer nested within dietary treatment was the random experimental error. Data were tested for normal distribution of the residues by the UNIVARIATE procedure of SAS (SAS/STAT release 9.1, SAS Institute Inc., Cary, NC). The data were assumed to have residues normally distributed when the Shapiro-Wilk statistic was equal to or greater than 0.90.
Count data, such as number of embryos-ova collected per heifer and number of cells per embryo, were analyzed by the GENMOD procedure by using a Poisson distribution and log-transformation function with the SAS program (SAS/STAT release 9.1). Continuous data with a normal distribution were analyzed by ANOVA with the GLM procedure of SAS (SAS/STAT release 9.1), with a model that included the effects of heifer treatment diet and the random experimental error.
Binary data, such as embryo hatching, compaction, and development into blastocysts, were analyzed by logistic regression with the LOGISTIC procedure of SAS (SAS/STAT release 9.1). Hours of development in vitro was analyzed by ANOVA with the GLM procedure of SAS (SAS/STAT release 9.1). In both cases, the mathematical model included the effects of heifer treatment (C, M, and H), embryo culture medium (CM and GM), the interaction between heifer treatment and embryo culture medium, and donor heifer nested within heifer treatment as the random experimental error.
Data are presented as least squares means and proportions. Treatment differences with P 
0.05 were considered significant and 0.05 < P < 0.10 were considered a tendency.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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), which resulted in similar concentrations of CP, NDF, and fat in the ration consumed by heifers (Table 1).
Gossypol Concentrations
Plasma gossypol concentrations increased (P < 0.001) as FG intake increased (Table 2). As expected, heifers fed C had almost undetectable TG in plasma. In fact, only 2 of the C heifers had detectable gossypol in plasma. Similar to TG, the concentrations of the (+) and the (–) isomers were greater (P < 0.001) for H than M, and for M than C heifers. Because Pima contains a slightly larger proportion of TG as the (–) isomer (Robinson et al., 2001; Santos et al., 2002, 2003), the (–) isomer in plasma usually increases to a greater extent than the (+) isomer (McCaughey et al., 2005; Santos et al., 2005), which might suggest that retention or metabolism of the 2 gossypol isomers differ. Because of such differences, heifers fed the M and H diets were expected to have more (–) than (+) isomer in plasma, as was observed previously (Santos et al., 2002, 2005; McCaughey et al., 2005).
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Of the first 20 mL infused in the uteri of heifers, a similar amount was recovered for each treatment (Table 2). The remainder of the fluid was left in the filter to ensure that embryos were not left without any flush solution. It is interesting to note that gossypol concentrations in the initial 20 mL of uterine flush infused and recovered paralleled those of plasma in heifers. Concentrations of gossypol in reproductive tissues of cows have not been described well. To our knowledge, only Velasquez-Pereira et al. (2002) reported gossypol concentrations in reproductive tissues of beef heifers consuming up to 16.5 mg of FG/kg of BW per d. The authors observed that both the endometrium and the CL had very high concentrations of TG (Velasquez-Pereira et al., 2002), indicating that gossypol accumulates in the reproductive tract of heifers. In fact, concentrations of gossypol in the endometrium ranged from 78 to 87 mg/kg of tissue, which were much greater than concentrations observed in plasma (Velasquez-Pereira et al., 2002). In the current study, uterine tissue was not collected, but uterine flush concentrations paralleled those of plasma gossypol concentrations, and heifers fed H had both plasma and uterine flush gossypol concentrations that have been shown to suppress embryonic development in vitro (Brocas et al., 1997). It is unknown whether gossypol concentrations in the uterine flush would remain constant throughout the embryo collection procedure or if the concentrations measured in the initial 20 mL of flush solution truly represent the quantities of gossypol that embryos are exposed to during their development in the uteri of cows fed cottonseed. Nevertheless, concentrations differed among treatments in a dose-response manner, which suggests that feeding FG increases TG concentrations in the uterine lumen, thereby exposing gametes and embryos to increased gossypol concentrations. Indeed, feeding increased amounts of FG to heifers increased plasma and follicular fluid gossypol concentrations in a parallel manner (Coscioni et al., 2003b).
Plasma Metabolites and BW
Because heifers receiving H had to be fed a large quantity of cottonseed to achieve the desired FG intakes, concentrations of CP and fat in the ration consumed (Table 1) were greater than typically recommended for this group of animals (NRC, 2001). Body weights throughout the study were similar among dietary treatments, averaging 406.1 kg (Table 3).
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). Concentrations of urea N, glucose, NEFA, and BHBA were similar (P > 0.10) for all treatments. Although not within the scope of this study, excessive intake of energy and protein can negatively affect embryo quality in cattle (Santos et al., 2008). Heifers fed protein in excess by adding urea to the ration had increased concentrations of urea N in plasma, reduced uterine pH, and reduced pregnancy per AI (Elrod and Butler, 1993). It is possible that embryo quality might not have been optimized in the current study because heifers had to be fed diets with increased nutrient concentrations to achieve the desirable FG intake. Nevertheless, dietary treatments resulted in similar nutrient intakes, BW, and plasma concentrations of metabolites.
Gossypol Intake on Superovulatory Response and Recovery of Embryos-Ova
Superovulatory response based on the number of CL observed by ultrasonography was less (P = 0.02) for heifers receiving the C diet than the M diet, but did not differ from those receiving the H diet (Table 4). Dietary FG intake did not influence the number of embryos-ova and embryos collected, and percentage of recovery. Coscioni et al. (2003b) evaluated superovulatory responses and recovery efficiency by using similar treatments and found no effect of gossypol intake on the number of ovulations and the number or proportion of embryos-ova recovered. Although the superovulatory response seemed to have been reduced in heifers fed C, the number of embryos-ova and only embryos collected and the proportion of recovery were not influenced by dietary FG.
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Dietary treatment did not affect the number of grades 1 and 2 embryos collected from heifers (Table 4). The number of degenerated embryos was similar between C and M heifers, but heifers fed H had an increased (P = 0.01) number of degenerated embryos. In fact, the numbers of unfertilized and low-quality embryos were greater (P = 0.03) for heifers fed H than for those fed C or M. Because gossypol alters lipid membrane conductance, which increases proton permeability, it can disrupt cell metabolism by altering the influx of ions into the cytoplasm and mitochondria of the cell (Reyes et al., 1984). When cows were fed gossypol, erythrocyte fragility increased (Velasquez-Pereira et al., 1998, 2002; Mena et al., 2004), which indicates changes in cell membrane integrity. Concentrations of gossypol in reproductive tissues were found to be higher than those commonly found in plasma when heifers were fed 14 mg of FG/kg of BW from cottonseed meal (Velasquez-Pereira et al., 2002). Such high concentrations of gossypol in reproductive tissues could result in disruption of cell membrane integrity in the early developing embryo, as has been demonstrated with erythrocytes. In fact, concentrations of gossypol in the plasma and uterine flush of heifers fed H were similar to those observed in the plasma of lactating cows with reduced pregnancy per AI (Santos et al., 2003), and of donor heifers that produced embryos with compromised ability to maintain pregnancy after embryos were transferred into lactating cows (Galvão et al., 2006). The changes in gossypol concentrations in reproductive tissues could account for the increased number of low-quality embryos-ova observed in this study when superovulated Holstein heifers were fed 36.8 mg of FG/kg of BW.
Gossypol Intake and Embryo Characteristics by Differential Staining
A total of 72 viable early morulae were stained immediately after collection, 23 from C, 28 from M, and 21 from H heifers. The grade quality of morulae from different treatments was similar before staining (Table 5). The numbers of cells were similar for C and M, but embryos from heifers fed H had fewer (P < 0.01) cells, primarily because of a reduced (P = 0.01) number of live cells. Furthermore, embryos from heifers fed H were smaller (P < 0.001) in diameter than embryos from heifers fed C and M.
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), which indicates that embryo development in vivo was impaired when heifers were fed 36.8 mg of FG/kg of BW.
Gossypol Intake and In Vitro Concentration of GAA on Embryo Development In Vitro
A total of 103 morulae were cultured in vitro, 29 C (15 CM, 14 GM), 35 M (19 CM, 16 GM), and 39 H (21 CM, 18 GM). Because no interaction (P > 0.20) between embryo donor dietary FG content and in vitro GAA concentration was observed in any of the outcomes evaluated, data are presented separately for the effects of dietary FG and in vitro gossypol concentrations (Table 6). Proportion of embryos compacting was not affected by dietary FG intake or in vitro incubation with GAA. Compaction of the embryonic cells, which occurs 5 to 6 d after fertilization, is a necessary developmental process to the formation of the blastocyst (Gordon, 1994). Such a process describes the formation of tight junctions between cells, which is necessary for the expansion of the blastocoele cavity through the accumulation of fluid, which is energy dependent (Van Soom et al., 1996).
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Although the presence of gossypol at 10 µg/mL tended to reduce the proportion of early morulae that reached the blastocyst stage, embryos in GM that developed to blastocyst reached that stage sooner (P = 0.02) than embryos from CM. Nevertheless, embryos cultured in GM developed for fewer (P = 0.02) hours in vitro than embryos cultured in CM. We did not anticipate that GM embryos would reach the blastocyst stage earlier than embryos cultured in CM. In any case, it was clear that both FG intake at 36.8 mg/kg of BW and the presence of gossypol at 10 µ/mL in the embryo culture medium compromised early embryo development.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication December 11, 2007. Accepted for publication March 27, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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