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Introduction
Susceptibility to oxidation increases during gestation and around birth (Castillo et al., 2005; Berchieri-Ronchi et al., 2011), and lipid oxidation can affect embryonic preimplantation (Guerin et al., 2001). As a result, multiple physiological processes such as oocyte maturation, fertilization, embryo development, and pregnancy can be affected (Agarwal et al., 2005). Several researchers have studied antioxidant supplementation during these stages in order to increase antioxidant capacity (Casamassima et al., 2012). A study by Salinas- Rios et al. (2016) found that antioxidant capacity decreases at the beginning of gestation. Thus, demand for antioxidants could increase prior to pregnancy and in early gestation.
Around 10 million tons of coffee residues are generated in the main producing countries (Echeverria and Nuti, 2017). Several studies have been conducted with the aim of using this residue in animal feeding (Lima de Souza et al., 2010; Salinas-Rios et al., 2015). In dairy cows, coffee pulp can be included up to 20% of the diet without affecting dry matter intake, milk production and composition, or body weight (Pedraza-Beltrán et al., 2012). However, little is known about the use of coffee pulp in reproduction. Considering its antioxidant compounds (Salinas et al., 2014), coffee residues have been tested prior to mating and during early gestation in sheep, finding that supplementation with 450 g concentrate with 25% coffee pulp decreased gestation. Additionally, no more losses are observed when supplementation is suspended. Coffee pulp contains 0.57% caffeine (Salinas et al., 2014) which could cause reproductive problems, since studies in women have reported that high caffeine intake reduces fertility (Fumihiro et al., 2005). However, Nawrot et al. (2003) reported that this effect on fertility is reduced for intakes lower than 300 mg/day. Caffeine exerts its effects only at the time it is delivered, and these effects disappear within 2 to 12 hours after consumption (Benowitz et al., 1990; Daly et al., 1993), so we hypothesized that reducing the amount of coffee pulp and suspending supplementation prior to mating could avoid embryonic losses.
Therefore, the aim of this work was to determine the oxidative status and reproductive efficiency of primiparous ewes supplemented with two levels of coffee pulp during estrus synchronization.
Materials and methods
Ethical considerations
The study was conducted at the experimental farm of Colegio de Postgraduados, Campus Montecillo, Mexico, under the rules of animal welfare, biosecurity and ethics of this institution, in compliance with the official Mexican norm NOM-062-ZOO-1999 (SAGARPA, 2001).
Experimental design and feeding
Sixty Dorset x Suffolk ewe lambs were used, with 48.66 ± 5.18 kg average weight and a body condition of 3 on a 1 to 5 scale. Ewes were distributed in a completely randomized design with three treatments of 20 animals each. A balanced diet according to the NRC (1985) requirements was offered (Table 1).
Dietary treatments were: T0 = without coffee pulp, T1 = with 5% coffee pulp, and T2 = with 10% coffee pulp. In order for all sheep to consume the same amount of feed, the average consumption was estimated for three days, which was 1.5 kg per animal. This amount was provided in two meals (8:00 am and 4:00 pm) for 16 days, starting two days before inserting the CIDR and until the moment of service.
Table 1 Composition of the diets fed to sheep during synchronization.
* Phosphorus 17.5%; Sodium 12.9%; Calcium 5.6%; Magnesium 3.4%. T0 = treatment without coffee pulp; T1 = treatment with 5% coffee pulp; and T2 = treatment with 10% of coffee pulp in the diet. DM = dry matter; CP = crude protein; and CF = crude fiber.
A sample of extract was obtained from the diet to determine antioxidant capacity, following the methodology described by Restrepo-Sánchez et al. (2009), making small modifications and using the FRAP (ferric reducing ability of plasma) from Benzie and Strain (1999).
Estrus synchronization
Pre-synchronization was carried out with two doses of 125 μg PGF2α (cloprostenol) at an interval of eight days. Six days after the second application, an intravaginal device impregnated with 0.3 g progesterone (CIDR®) was inserted and kept for 11 days. Twelve hours after CIDR withdrawal, estrus was detected with males fitted with aprons at 6 h
Figure 1 Estrus synchronization protocol and sampling to measure oxidative status, progesterone, glucose and insulin in sheep supplemented with coffee pulp. CIDR: vaginal progesterone-releasing device; PGF2α: prostaglandins; TBARS: reactive substances to the thiobarbituric acid; FRAP: antioxidant power of ferric reduction. Oxidative state
To determine antioxidant capacity and lipid oxidation, nine samples were collected every third day, starting two days before the supplementation and until breeding (Figure 1). Blood samples were obtained from the jugular vein from fasted ewes at 8:00h in 5 ml vacutainer tubes with EDTA (ethylenediaminetetraacetic acid). Samples were centrifuged at 2,500 rpm for 20 minutes at -4°C (SIGMA 2-16 PK SIGMA Laborzentrifugen Gmbh, Osterode am Harz, Germany). Plasma was aliquoted in 1.5 ml Eppendorf tubes and stored at -40°C until further analysis. For antioxidant capacity determination, FRAP from Benzie and Strain (1999) was used, and for lipid oxidation the technique by Ohkawa (1979) was employed.
Progesterone (P4), glucose and insulin
Eight samples were collected to determine P4, and three for glucose and insulin at the beginning, middle and at the end of the dietary supplementation (Figure 1). Progesterone determination was done by an immunoenzymatic assay (Immunometrics, UK Ltd, 280 Muster Road, London SW6 6BQ) with 0.13 ng mL-1 analytical sensitivity, and intra- and inter-assay coefficient of variations of 9.59 and 13.7%, respectively. Plasma insulin analysis was performed by Radioimmunoassay (RIA) with 4.09 ng mL sensitivity, and intra- and inter-assay coefficients of variations of 1.44 and 0.25%, respectively. Glucose determinations were performed by the enzymatic method based on glucose oxidase (Pointe Scientific, Inc, USA.).
Statistical analysis
The number of offspring and gestation rate (%) were analyzed under the procedure PROC FREQ with the chi-square test. For percentage of births, birth weight, onset and duration of estrus a Tukey mean comparison tests were applied using PROC GLM procedure of SAS® software, Version 9 (SAS Institute Inc, Cary, NC, USA) (2002).
Antioxidant capacity, lipid oxidation, progesterone, glucose and insulin variables were tested for normality and homogeneity of variances and analyzed under the test of repeated measurements over time using PROC MIXED. The statistical model was:
Where:
Yijk= is the response of the i-th treatment in the j-th sampling of the k-th repetition, μ= is the general mean, Ti= is the effect of i-th treatment, Mj = is the effect of j-th sampling, TiMj= is the effect of i-th treatment in j-th sampling, Ak (i)= Effect of the i-th treatment nested on the k-th animal, Eijk= Experimental error. A Tukey test was performed for the variables that showed a significant difference (p<0.05).
Results
Antioxidant effect of diets
Antioxidant effect of diets fed to the animals increased when the level of inclusion of coffee pulp was higher. T1, T2, and T3 resulted in 45.18, 51.55, and 137.02 μmol trolox/g DM, respectively (Table 2).
Table 2 Antioxidant capacity of diets fed to ewes during estrus synchronization.
T0 = treatment without coffee pulp; T1 = treatment with 5%; and T2 = treatment with 10% of coffee pulp in the diet. FRAP: ferric reducing-antioxidant power.
Table 3 Reproductive variables of synchronized ewes supplemented with coffee pulp.
T0 = treatment without coffee pulp; T1 = treatment with 5% and T2 = treatment with 10% inclusion of coffee pulp in the diet. No statistical differences were found (p>0.05).
Reproductive variables
After CIDR withdrawal, 100% of the ewes presented estrus. Coffee pulp inclusion for the 5 and 10% diets did not modify the onset or duration of estrus, neither the gestation percentage at 30 and 60 days, nor the number of offspring or birth weight (p>0.05) (Table 3).
Antioxidant capacity in blood
The antioxidant capacity of blood plasma increased (p<0.05) as the percentage of coffee pulp inclusion increased, being greater in ewes fed 10%, followed by supplementation with 5%, and, finally, the control treatment (Table 4).
Table 4 Antioxidant capacity and lipid oxidation in ewes fed different inclusion levels of coffee pulp during estrus synchronization.
Different superscript letters (a, b, c) within the same column indicate statistically significant differences (p<0.05). T0 = treatment without coffee pulp; T1 = treatment with 5%; and T2 = treatment with 10% inclusion of coffee pulp in the diet. MDA: malondialdehyde; SEM: standard error of the mean.
During sampling, antioxidant capability decreased after CIDR withdrawal (Figure 2).
Figure 2 Antioxidant capacity in ewes during estrus synchronization with progestogen. CIDR: vaginal progesterone-releasing device; PGF2α: F2α- prostaglandin; Trolox: trolox equivalent antioxidant capacity. Different letters (a, b, c) indicate statistically significant differences between bars (p<0.05). Lipid oxidation
Inclusion of 10% coffee pulp in the diet reduced (p<0.05) lipid oxidation in plasma with respect to control ewes. There was no statistical difference (p>0.05) between inclusion of 10 and 5%; nevertheless, as coffee pulp inclusion increased lipid oxidation in the blood decreased (Table 4). Regarding the date of sampling, there was no clear effect of lipid oxidation through sampling. Lowest values (p<0.05) were found between experimental days 9 to 15 (corresponding to 5 days before CIDR withdrawal and 5 days after) with respect to days 1, 5, 7 and 17 (Figure 3).
Figure 3 Lipid oxidation in ewes during estrus synchronization with progestogen. CIDR: vaginal progesterone-releasing device, PGF2α: F2α-prostaglandin. MDA: malondialdehyde. Different letters (a, b, c) indicate statistically significant differences between bars (p<0.05). Progesterone (P4)
There were no statistical differences (p>0.05) in P4 concentration with the inclusion of 5 to 10% of coffee pulp during estrus synchronization. Regarding sampling date, a higher concentration of progesterone (p<0.05) was observed when ewes had the CIDR (experimental day 8), decreasing after withdrawal, followed by a gradual increase from experimental day 18, corresponding to one day after mating (Figure 4), and maintaining a normal secretion profile.
Figure 4 Concentration of progesterone in ewes during estrus synchronization with progestogen and beginning of pregnancy. CIDR: vaginal progesterone-releasing device; PGF2α: F2α-prostaglandin. Different letters (a, b, c, d, e, f, g) indicate statistically significant differences between bars (p<0.05).
Glucose and insulin concentration in blood serum
Inclusion of 5 and 10% coffee pulp in the diet did not modify (p>0.05) the content of glucose and insulin. A higher concentration of glucose and insulin (p<0.05) was observed in all treatments in the last two samples, which was the period when animals were supplemented with coffee pulp (Table 5).
Discussion
The antioxidant compounds present in coffee pulp (Arellano et al., 2011; Murthy and Naidu, 2012) increase antioxidant capacity. Oat hay was used as a source of forage, and the 5% replacement for coffee pulp slightly increased antioxidant capacity; however, the inclusion of 10% increased more than three times the antioxidant capacity, so we suggest that inclusion of pulp is feasible for diets with low amounts of antioxidants, since little of this by-product is needed to improve the antioxidant capacity of the diet.
Reproductive performance
Inclusion of pulp for a short period prior to breeding did not affect the reproductive variables. In a previous study (Salinas-Rios et al., 2016), we found that supplementing with 450 g of concentrate with 25% of coffee pulp (corresponding to a coffee pulp consumption of 112.5 g per day) for 25 days before and 25 days after CIDR withdrawal resulted in decreased gestation percentage, which was attributed to the caffeine present in coffee pulp. If ewes consumed 1.5 kg feed, then pulp consumption was 75 g for the treatment with 5% inclusion, and 150 g for the treatment with 10%. On average, 5% inclusion corresponds to a lower level, and 10% at a higher level than previously offered (Salinas-Rios et al., 2016); therefore, we assume that up to 10% pulp supplementation for a short period is feasible, although the maximum inclusion should be evaluated.
Adverse effects of coffee pulp on reproduction have been reported to disappear when supplementation is suspended. Salinas-Rios et al. (2016) did not observe more reduction in gestation rate after finishing supplementation. In the present study, reproductive variables were not affected when supplementation was provided during estrus synchronization. In this regard, Natarajan et al. (2007) indicated that caffeine half-life is 103 hours in plasma.
Caffeine was previously reported as not reducing fertility in women (Wesselink et al., 2016); According to Valenzuela (2010), pregnant women consuming less than 300 mg of caffeine do not present risks. A study with pregnant women in which one group was given decaffeinated and the other caffeinated coffee concluded that caffeine intake reduction starting in mid-gestation had no effect on the duration of pregnancy or birth weight (Molina, 2007). According to these data, the damage that caffeine could cause depends on the dose consumed. Coffee pulp contains 0.57% caffeine (Salinas et al., 2014), so consumption of 150 g of coffee pulp in the treatment with 10%
supplementation would be equivalent to a consumption of 0.855 g caffeine, although part of this caffeine could be degraded in the rumen.
Oxidative state
The antioxidant capacity in blood plasma was greater when coffee pulp increased; whereas malondialdehyde concentration followed an inverse tendency, indicating that antioxidants in coffee pulp help reduce lipid peroxidation. Gupta et al. (2007) reported an increase in MDA, and therefore oxidative stress, causing placental dysfunction and the repercussions originated by this disorder, such as recurrent pregnancy loss, defective embryogenesis, among other problems; thus, during this disorder there is high antioxidant demand and antioxidant capacity decreases (Kankofer et al., 2010). Recent studies have shown the ability of some plants to counteract lipid oxidation; Pérez-Hernández et al. (2013) added cranberry extract (Vaccinium meridionale SW) to 50 g corn oil observing that lipid oxidation decreased; likewise, Londoño et al. (2013) evaluated the antioxidant activity of different types of coffee by checking their ability to prevent low density lipoprotein (LDL) oxidation. Furthermore, Zhang et al. (2011) found that adding antioxidants to hen diets increases the incubation ability of fertile eggs, increasing the number of hatchings. Benefits of improving the oxidative status in animals have been reported; for example, Sönmez et al. (2009) found that vitamin E prevents overproduction of free radicals, thereby preventing oxidation and increasing the number of multiple births. Placental retention is also reduced and improvement in the immune system has been observed (Spears and Weiss, 2008). In addition, improved antioxidant capacity in lambs has been reported by supplementing their mothers with antioxidants (Nieto et al., 2010); however, in the present study, reproductive benefits were not observed by improving the antioxidant status in ewes, probably due do the pregnancy rate in the control group being 90%, which is high. However, benefits of coffee pulp during oxidative stress -when antioxidant requirement increases- should be evaluated.
Progesterone
Coffee pulp does not affect P4 concentration in primiparous ewes. The highest progesterone concentration was reached when the animals had the CIDR combined with the corpus luteum present at that moment in the ovary. Accordingly, supplementing coffee pulp for a short period prior to pregnancy would not have a negative effect on this hormone; progesterone is the main hormone responsible for maintaining pregnancy, and low concentrations at the beginning of gestation could trigger abortions (Diskin and Morris, 2008); its increase is, therefore, associated with improved gestation rate (Flores et al., 2013).
Glucose and insulin
Glucose and insulin concentrations were not affected by coffee pulp inclusion in the diet. Glucose concentration was 64.42 mg/dl during estrus synchronization. These results are similar to those reported by Balaro et al. (2015) who found an average concentration of 64.8 mg/dl in sheep under tropical conditions. The optimal glucose range is between 50 and 80 mg/dl (Kaneko, 1980), hence, all treatments in all samples were within normal range.
Insulin concentrations in this study ranged from 0.39 to 0.51 ng/ml between treatments during estrus synchronization. It has been reported that concentrate supplementation increases insulin and glucose concentrations (Razz and Clavero, 2004; Noro et al., 2006; Catunda et al., 2013). This agrees with our results; although coffee pulp did not modify glucose and insulin concentrations in ewes, they increased when animals consumed the concentrate. In our previous study (Salinas et al., 2015) we found that feeding coffee pulp to sheep during fattening reduces fat in the rumen and intestines which is not desirable in pregnant sheep, since, during this time the female accumulates fat for lactation. This fat mobilization could affect the metabolic state of the animal. In this regard, Mahmoud and Azab (2014) reported that glucose and insulin concentrations vary throughout pregnancy and lactation in goats, so it is assumed to be a normal mechanism for the development of the embryo and fetus, and the physiological processes occurring during those stages. Caffeine intake can alter glucose metabolism (Du et al., 2006), so coffee may contain compounds that increase glucose absorption and decrease insulin. Bilge et al. (2017) gave instant coffee to people and measured metabolic hormones in blood three hours post consumption finding no change in glucose or insulin concentration. Van Dam et al. (2004) reported that glucose metabolism could be modified during coffee ingestion. Glucose and insulin concentrations may play a crucial role in pregnancy. During this period there is an increase in glucose levels, necessary for fetus survival, who obtains this metabolite from the mother (Herrera, 1997). This study found that coffee pulp at the dose and time supplied does not alter glucose or insulin levels.
Our results indicate that up to 10% coffee pulp can be included in the diet of ewes for 16 days prior to breeding with no detrimental effects on pregnancy, estrus or prolificacy, as well as progesterone, glucose or insulin concentrations. Additionally, coffee pulp improves the oxidative status of the animal, which merits further investigation about its potential benefits on the reproductive ability of sheep.
