Inﬂuence of Copra Meal in the Lambs Diet on In Vitro Ruminal Kinetics and Greenhouse Gases Production

: The present study aimed to evaluate the effect of copra meal (the waste coconut of the oil industry) on in vitro ruminal kinetic and greenhouse gases production and on in vivo lamb performance. Twenty-eight male Rambouillet sheep (initial body weight 24.5 ± 3.9 kg) were randomly assigned to one of the four treatments: 0, 50, 100, and 150 g of copra meal/kg in their diet (dry matter basis). Final weight, weight gain, and feed intake were not affected ( p > 0.05) by the copra meal addition. The gas production volume (V) decreased, and the gas production rate increased, in a linear trend ( p < 0.05) as copra meal was added to the diet. In contrast, methane and CO 2 production showed an opposite quadratic trend ( p < 0.05), with the highest and lowest values reported at 100 g/kg DM of copra meal, respectively. The addition of copra meal in the lambs’ diet decreases the volume of gas production and is a strategy to decrease methane and carbon dioxide production in feeding without affecting animal performance.


Introduction
Sheep, like other ruminants, use fibrous materials and convert them into high nutritional value products (meat and milk) [1]. However, in tropical countries, due to climatic seasonality conditions, the availability and quality of forage resources are really variable [2]. Therefore, animal feed is based on agro-industrial waste available in different regions of the world, which constitutes a viable alternative from a productive and economic perspective [3]. Copra meal is one of the alternative by-products produced mainly in some tropical areas of the world [4]. Copra meal is obtained from the solid part of the endosperm of the nut and is reduced to pieces. It is usually considered waste, and it is mentioned that large quantities of this are left to rot in the fields as unwanted material [5]. Some reports have calculated an energy value equivalent to 2.5 kg of corn and sorghum, which makes it attractive for ruminants feed due to its energy value, 4.7 Mcal/kg DM (Dry Matter). In this sense, González-Garduño et al. [6] reported that supplementation with coconut paste increases weight gains in lambs at grazing; these results may be due to high oil content.
Methane (CH 4 ) and carbon dioxide (CO 2 ) are potent greenhouse gases [7]. These gases contribute 14.3% and 73.9%, respectively, to anthropogenic global greenhouse gases [7]. In addition, 17% to 37% of global anthropogenic methane (CH 4 ) emissions have been estimated to result from ruminant livestock [8], and practices related to livestock production are estimated to produce around 10.8% of the global greenhouse emissions [9,10].
Other research has reported that the use of plant oils reduces enteric CH 4 emissions via ruminal defaunation [11], toxic effects on methanogens [12], and decreased diet digestibility [13]. Jordan et al. [12] identified that 250 g of refined coconut oil/d fed to beef heifers (approximately 500 kg of BW) could reduce enteric CH 4 emissions and positively affect the fatty acid profile in the muscle produced in terms of human nutrition health. High quantities of coconut oil can be retained in copra meal if the industrial oil extraction process is inefficient [12]. Jordan et al. [13] observed that coconut oil in copra meal might influence ruminal fermentation and promote a reduction in methane production, as was observed when refined coconut oil was used in diets [11,13]. Thus, the current study aimed to evaluate the effect of copra meal on in vitro ruminal kinetic and carbon dioxide and methane gas production.

Productive Phase
The experimental trial was conducted at the lambs experimental and production unit of UASLP, Mexico. Four experimental diets (Table 1) of 0, 50, 100, and 150 g of copra meal kg −1 DM were randomly assigned to 28 Rambouillet lambs (initial body weight 24.5 ± 3.9 kg) housed in individual cages in a naturally ventilated barn. Balanced diets were according to the nutritional requirements by NRC [14]. The diets were provided at 08:00 and 15:00 h each day. At the beginning of the experiment, lambs were adapted to the diets for seven days; experimental investigation began after this period and lasted for 42. Feed intake and orts were measured daily. Every 14 days, lambs were weighed before their morning feeding. The feed conversion was calculated as the ratio of daily intake to daily weight gain. Samples of the experimental diets were taken every 14 days. The experimental diets were analyzed for their chemical composition and dry matter (DM), crude protein (PC) according to the AOAC [15], neutral detergent fiber (NDF), and acid detergent fiber (ADF) [16].

In Vitro Gas Production
Experimental diets were used as substrates for in vitro incubations formulated in an isoproteic and iso-energetic manner. On the other hand, a sample of 100 g from each diet was taken for its later bromatological analysis. The samples were dried at 65 • C for Agriculture 2021, 11, 925 3 of 7 24 h. Once dried, the samples were milled to a sieve with a mesh 1 mm in diameter. For incubation, 500 mg of each diet sample was incorporated into 90 mL of culture medium.
The reduced (without oxygen) culture media per liter used in vitro contained (Cobos and Yokoyama [17]): mineral solution A, 6 g of potassium hydrogen phosphate was added to 1 L of distilled water; mineral solution B, 6 g of potassium hydrogen phosphate, 1.6 g calcium chloride, 6 g ammonium sulfate, 12 g sodium chloride 2.45 g magnesium sulfate, and 18% sodium carbonate buffer solution, were added to 1 L of distilled water; and a cysteine reduced solution, where 2.5 g of sodium sulfide and 0.1 mL of resazurin (1%) was mixed with mineral solutions and CO 2 pumped until the marker turned.
Two Rambouillet rams, fed a diet of 50% corn grain plus 50% corn silage, were ruminal fluid donors. The ruminal fluid (10 mL) and the culture media (90 mL) were added to the amber flask [18] that contained 500 mg of the diet. These samples were incubated at 39 • C in a water bath. Ten repetitions of each diet and three blanks for correction were used.

In Vitro Dry Matter Degradability (IVDMD)
At the end of incubation (96 h), each Ankom bag (n = 5) was washed and then dried for 48 h at 65 • C to calculate the level of DM disappearance.

Carbon Dioxide (CO 2 ) and Methane (CH 4 ) Estimation
Sequential selection of each tube's outflow to an infrared gas analyzer (IRGA) attached to a custom chamber (Li-6400XT and 6400-09, LiCor, Lincoln, NE, USA) allowed for the monitoring of CO 2 mixing ratios in real-time. Later, the production levels of CO 2 , at 4, 8, 12, 24, 36, 48, and 72 h of fermentation, were measured. Estimations of the methane levels were developed through the analysis of the proportions in these measured CO 2 values [20] and the time that this proportion was calculated [21].

Statistical Analysis
The data obtained were analyzed using a completely randomized design [22]. Orthogonal polynomial contrasts were used to verify linear or quadratic effects for the copra meal levels. The PROC NLIN BEST method (SAS Institute, 2002, NC, USA) was performed to calculate the ruminal in vitro kinetics (volume of gas produced, gas production rate, and initial gas production). Data were analyzed with JMP7 software [23]. A p-value of 0.05 was selected as the significance level.

Productive Test
The effect of copra meal on final weight, dry matter intake, average daily gain, and feed conversion was not statistically significant. The lambs showed similar growth performance with fed diets of 0, 50, 100, and 150 g ( Table 2). Final weight, weight gain, and feed intake were lower in lambs whose diets 100 g/kg MS of copra meal than in other treatment conditions.

In Vitro Dry Matter Degradability (IVDMD)
A quadratic trend (p < 0.05) was found for the in vitro digestibility of dry matter (IVDMD) between treatments at 0 h and 48 h; however, at 96 h IVDMD, a tendency was not detected (Figure 1).
The data obtained were analyzed using a completely randomized design [22]. Orthogonal polynomial contrasts were used to verify linear or quadratic effects for the copra meal levels. The PROC NLIN BEST method (SAS Institute, 2002, NC, USA) was performed to calculate the ruminal in vitro kinetics (volume of gas produced, gas production rate, and initial gas production). Data were analyzed with JMP7 software [23]. A p-value of 0.05 was selected as the significance level.

Productive Test
The effect of copra meal on final weight, dry matter intake, average daily gain, and feed conversion was not statistically significant. The lambs showed similar growth performance with fed diets of 0, 50, 100, and 150 g ( Table 2). Final weight, weight gain, and feed intake were lower in lambs whose diets 100 g/kg MS of copra meal than in other treatment conditions.

In Vitro Dry Matter Degradability (IVDMD)
A quadratic trend (p < 0.05) was found for the in vitro digestibility of dry matter (IVDMD) between treatments at 0 h and 48 h; however, at 96 h IVDMD, a tendency was not detected (Figure 1).

In Vitro Gas Production
Gas production volume (Vm) was affected in a linear way by the addition of copra meal (p < 0.05). Moreover, the gas production rate (s) increased linearly (p < 0.05) as the amount of copra meal increased. The lag phase (L) showed no difference (p < 0.05) between treatments ( Table 3). The production of methane presented a quadratic effect (p < 0.05), having a maximum production value (34.03%) in the diet with 100 g/kg MS of copra meal. In the case of carbon dioxide, a quadratic effect (p < 0.05) was also registered, but with a minimum value (65.96%) in the diet with 100 g/kg MS of copra meal. Although no differences (p < 0.05) were observed for the values of short-chain fatty acids (SCFA) or metabolizable energy (ME), a numerical increase in metabolizable energy was observed for the diet with 100 g/kg MS of copra meal (Table 3).

Productive Test
Copra meal had no effect on the final weight, dry matter intake, average daily gain, or feed conversion of the lambs (Table 2). This effect could be comparable with the results of beef heifers supplemented with copra meal by Jordan et al. [12]. Jordan et al. [12,13] did not observe changes in food consumption when maintaining the diet at isotropic and iso-energetic levels. Jordan et al. [13] concluded that the amounts of other ingredients (alfalfa, sorghum, soybean meal) are modified, and the animals could reach a steady state. Although a difference in production parameters was not observed, the inclusion of 100 g/kg MS of copra meal in diets showed the lowest weight gains. In contrast with our results, Jordan et al. [12] reported an improvement for these variables. We theorized that the improvements could be attributed to maintaining the same level of consumption, as the oil content would increase the animal's energy content and decrease energy loss in the form of methane [24].

In Vitro Dry Matter Degradability (IVDMD) and In Vitro Gas Production
A quadratic effect was observed for IVDMD during 0 h to 48 h. However, at 96 h, no changes were detected ( Figure 1). Lima et al. [25] observed that the in vitro digestibility of organic matter ranged from 35.91 to 61.18% in diets of 0 to 30% copra meal. The maxi-mum degradability was found among the control group, coinciding with the values found in this research. The fat content in copra meal affects digestibility because it forms a barrier between the ruminal microorganisms and feed particles [26].
Copra meal increases linearly with the gas production volume and rate ( Table 3). The diet that contained 100 g/kg MS showed a maximum gas production of 188.7 mL g −1 , a value higher than in any other treatment condition. However the treatment with 150 g kg −1 MS showed the minimum gas production level of 153.2 mL g −1 , thus confirming the potential of coconut oil to inhibit ruminal fermentation, as has been previously recognized [25]. Hollmann and Beede [27] mention that diets with 2.5% coconut oil decreased milk production, correlating with decreased dry matter digestibility and neutral detergent fiber.
The production of methane showed a quadratic reduction in its gas production value in the diet with 100 g/kg MS of copra meal. Similarly, the copra meal reduced the carbon dioxide gas production value when diets with 100 g/kg MS were used. A reduction in methane production as copra meal increased could be explained through the highfat content diets that reduce methanogenesis by inhibiting the activity of protozoa and archaeans. These diets also inhibit the biohydrogenation of unsaturated fatty acids that act as hydrogen receptors [28,29]. Soliva et al. [30] demonstrated that garlic oil is more effective in mitigating methane production without decreasing nutrient utilization efficiency. O' Brien et al. [31] indicated that the use of saturated fatty acids could be an efficient strategy to mitigate methane production from inhibition in in vitro ruminal fermentation, considering the diet-dose correlation. It could also be considered to mitigate carbon dioxide production by enteric digestion, as reveal the results of this research.

Conclusions
The addition of copra meal to a diet, under in vitro conditions, decreases the gas production volume equal to that of methane and carbon dioxide production. However, it does not affect the lamb's performance.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest in the development of this manuscript or in the decision to publish the results.