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Supplementation of diets with omega-3 fatty acids from microalgae: Effects on sow reproductive performance and metabolic parameters
Author’s Accepted Manuscript Supplementation of diets with omega-3 fatty acids from microalgae: effects on sow reproductive performance and metabolic parameters C.J.M. Posser, L.M. Almeida, F. Moreira, I. Bianchi, B.G. Gasperin, T. Lucia www.elsevier.com/locate/livsci
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S1871-1413(17)30333-5 https://doi.org/10.1016/j.livsci.2017.11.006 LIVSCI3344
To appear in: Livestock Science Received date: 7 March 2017 Revised date: 25 October 2017 Accepted date: 2 November 2017 Cite this article as: C.J.M. Posser, L.M. Almeida, F. Moreira, I. Bianchi, B.G. Gasperin and T. Lucia, Supplementation of diets with omega-3 fatty acids from microalgae: effects on sow reproductive performance and metabolic parameters, Livestock Science, https://doi.org/10.1016/j.livsci.2017.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supplementation of diets with omega-3 fatty acids from microalgae: effects on sow reproductive performance and metabolic parameters C.J.M. Posser1, L.M. Almeida2, F. Moreira1, I. Bianchi3, B.G. Gasperin1, T. Lucia Jr. 1* 1
ReproPel, Faculdade de Veterinária, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil 2
Faculdade Evangélica do Paraná, 80730-000, Curitiba-PR, Brazil 3
Instituto Federal Catarinense, 89245-000, Araquari-SC, Brazil
Abstract Supplementation of diets with omega-3 polyunsaturated fatty acids (PUFA) may benefit sow reproductive performance, but the efficiency of algae as a source of PUFA is still unknown. This study evaluated serum levels of metabolic markers and reproductive performance of sows supplemented with the heterotrophic microalgae Schizochytium sp. Sows (n = 596) were supplemented from the 85th day of gestation, during lactation and until the weaning-estrous interval (WEI). Microalgae were included at five levels: 0 (control); 3.5; 7.0; 14.0; and 28.0 g/d. Blood samples were collected at the 85th day of gestation, at the 10th d of lactation and at the last day prior to weaning (WEI). Serum levels of cholesterol, triglycerides, insulin-like growth factor type-1 (IGF-1) and nonesterified fatty acids (NEFA) were evaluated. At the parity concurrent with the supplementation, no effects were observed on stillborn rates (P > 0.05), sows fed 28.0 g/d farrowed heavier piglets than sows fed lower levels (P < 0.05), but the WEI for sows fed 7.0 g/d was longer than for the control (P < 0.05). Serum levels of triglycerides were decreased during gestation, only for sows supplemented with 14.0 and 28.0 g/d (P < 0.05). At the subsequent parity, there was no difference on stillborn rates and total Author’s address (for correspondence): T. Lucia Jr., ReproPel, Faculdade de
*
Veterinária, Universidade Federal de Pelotas, Pelotas, RS 96010-900, Brazil. E-mail: [email protected]
litter size (P > 0.05). The highest tested concentration (28.0 g/d) of Schizochytium sp. was associated with reduced serum levels of triglycerides during gestation and with increased piglet birth weight, but the supplementation generally did not benefit sow reproductive performance. Key words: DHA, litter size, stillbirths, birth weight, metabolism, sows.
1. Introduction Total litter size has increased over time in swine due to genetic selection for prolifacy, concurrently with an increase in stillborn rates and in the number of piglets born with low viability (reviewed by Prunier et al. 2010). Thus, strategies to improve post-natal piglet development are of interest to the swine industry. Gestation and lactation diets may be supplemented with polyunsaturated fatty acids (PUFA), especially those of the omega-3 series (reviewed by Rossi et al. 2010). Omega-3 PUFA can be incorporated into oocytes, as observed for cattle and sheep (Zachut et al. 2008; Gulliver et al. 2012) and into tissues of swine embryos and fetuses (Brazle et al. 2009), potentially benefiting their subsequent development. Omega-3 PUFA may also affect metabolic markers, such as triglycerides, non-esterified fatty acids (NEFA), insulin-like growth factor type 1 (IGF-1) and cholesterol, influencing energy metabolism and the function of the reproductive axis (Prunier and Quesnel 2000; Harris and Bulchandani 2006; Adkins and Kelley 2010). Despite of reported benefits on fetal development (Innis 1991) and on litter size (Smits et al. 2011), effects on fetal survival may be inconsistent (Perez Rigau et al. 1995). Thus, the effects of supplementation with omega-3 PUFA on sow performance are still controversial, especially due the variety of their available sources.
The docosahexaenoic acid (DHA) is one of the most important omega-3 PUFA (reviewed by Rossi et al. 2010; and by Tanghe and de Smet 2013). The DHA may have several sources, including vegetable (Ryckebosch et al. 2012), fish (Moreira et al. 2016) and marine oils (Smit et al. 2013; 2015). Fish oils are the most commonly used sources, but their use may be questioned due: low availability; inconsistent nutritional profile; risk of contamination by chemicals or pollutants deposited at the sea; and sustainability issues (Ryckebosch et al. 2012). On the other hand, omega-3 PUFA extracted from algae are non-pathogenic and nontoxigenic (Martins et al. 2013), representing a lowcost sustainable source with stable nutritional levels, high lipid content and greater DHA content than vegetable oils (Ryckebosch et al. 2012; Martins et al. 2013). Nevertheless, effects of supplementation with PUFA from algae on metabolism and reproductive performance of sows are not yet known. The objective of the present study was to evaluate the effects of supplementing gestation and lactation diets with heterotrophic microalgae on sow’s reproductive performance and metabolic parameters.
2. Materials and methods 2.1. Experimental design The study was conducted in a commercial farm with an inventory of 3,400 F1 crossbred females (PIC Camborough®) located in southern Brazil (latitude 27°22’35”S, longitude 51°02’38”W). All experimental procedures were approved by the Ethics in Animal Experimentation Committee of the Universidade Federal de Pelotas. The study was designed to include 650 sows. In each weekly group, sows were randomly assigned to five treatments (n = 130 sows each). Such sows had no locomotion problems and were neither used as nurses during lactation, nor previously programmed to be culled after lactation. Thereafter, 54 sows were censored: those that had unplanned culling
during the experiment; and those with unreliable records for dates of WEI and artificial inseminations. Sows were not paired by parity within treatments, due to logistic constraints of the farm’s production flow. The parity distribution of the remaining 596 sows was as follows: 2 (n = 149); 3 (n = 128); 4 (n = 104); 5 (n = 94); and 6-7 parities (n = 121). A commercial source of heterotrophic microalgae Schizochytium sp. (All-GRich, Alltech Inc.) containing 120 g/kg DHA (Supplementary material) was added to the diets, during the last 30 d of gestation and the lactation (21.9 ± 0.9 d), until the weaning-estrous interval (WEI) (3.5 ± 0.8 d). The supplement was a seaweed meal which was top-dressed to the diet on an individual basis, to guarantee that all sows were fed the same amount of the product. The treatments represented five supplementation levels: 0 (control, n = 121); 3.5 (n = 123); 7.0 (n = 114); 14.0 (n = 123); and 28.0 g/d (n = 115). During gestation, sows were kept in individual crates with automatic drop feeders, receiving 2-3 kg/d of a gestation diet containing: 14.4% crude protein; 3,100 Kcal/kg ME; and 3.2% fat. The amount of the diet was adjusted to each sow’s body condition. Prior to the predicted farrowing date (2-3 d), sows were housed at farrowing rooms, with ad libitum access to feed after the 4th d of lactation. The lactation diet contained: 20.1% crude protein; 3,337 Kcal/kg ME; and 4.8% fat. After weaning, sows were kept in individual crates and submitted to flushing with the lactation diet, until detected in estrous. Estrous detection was done conducted once a day, through back pressure in the presence of a sexually mature boar. Artificial insemination was done at 24 h-intervals, via intracervical, with 3 x 109 viable spermatozoa (2.4 AI per sow, on average).
2.2. Metabolic analyses From 45 sows selected at random within the same weekly weaning group (n = 9 per treatment), blood samples were collected from the jugular vein at: the 85th d of gestation; the 10th d of lactation; and the last d before weaning. After centrifugation at 7,000 x g for 5 min, the serum was placed inside cryotubes and stored in liquid nitrogen. Serum levels of cholesterol (11539, BioSystems, Curitiba, PR, Brazil; sensitivity 0.3 mg/dl) and triglycerides (87.1/250, Labtest, Lagoa Santa, MG, Brazil; sensitivity 3.0 mg/dl) were evaluated through standard enzymatic procedures. Chemiluminescence was used to assay free IGF-1, after acid treatment to allow precise quantification (Siemens Immulite 2000 IGF-1 L2KGF2: Siemens Healthcare Ltda, São Paulo, SP, Brazil; sensitivity 20.0 ng/mL). Serum levels of NEFA were evaluated by spectrophotometry (NEFA-HR2 Wako, Neuss, Germany; sensitivity 1.0 mEq/L). All assays were conducted in duplicate, in a commercial laboratory, using kits developed for human serum, but with multi-species validation (all CV inferior to 10%). 2.3. Reproductive performance At the parity concurrent to the supplementation, the number of total pigs born and stillborn was recorded at farrowing and the WEI was recorded. Stillborn rates per farrowing were calculated by dividing the number of stillborns in each litter by the total litter size, to adjust such rate for total litter size. For 55 randomly selected farrowings, individual piglet birth weights were determined using a computerized scale (Urano® US 20/2 pop light). The sex of the weighted piglets was also recorded. At the subsequent parity, total litter size and stillborn rates per farrowing were also recorded.
2.4. Statistical analyses Total litter size, stillborn rates and WEI were compared among treatments and parities through analyses of variance. As stillborn rates and WEI were not normally distributed, such variables were submitted to an angular transformation (arcsine of the square root of each value/100). Piglet birth weight at the parity concurrent to the supplementation was compared among treatments, with adjustment for the following covariates: parity of the sow; total litter size; and sex of each piglet. Serum levels of cholesterol, triglycerides, IGF-1 and NEFA were compared across treatments and periods of sample collection by analyses of variance with repeated measures, considering parity as a random factor and the individual effect of sows nested within the effect of period. Due to lack of normality, serum cholesterol levels were transformed to the logarithmic scale. In all models, comparisons of means were conducted using Tukey test. All analyses were conducted using Statistix® (2014).
3. Results No significant treatment per parity interaction affected the WEI at the parity concurrent with the supplementation and stillborn rates at both tested parities (P > 0.05). The WEI for sows supplemented with 7.0 g/d microalgae was longer (P < 0.05) than for control group (Table 1). Across parities, WEI was longer (P < 0.05) for sows with 2 (3.6 ± 0.1) and 6-7 parities (3.7 ± 0.1) than for sows with 3 and 4 parities (3.4 ± 0.1 and 3.3 ± 0.1, respectively). At the parity concurrent with the supplementation, total litter size was 14.4 ± 3.5, and born alive litter size was 13.4 ± 3.4. The percent of stillbirths was not influenced (P > 0.05) by the treatments (Table 1). Stillborn rates for sows with 6-7
parities (6.6 ± 0.7) were greater than at lower parities (P < 0.05), which ranged from 4.2 ± 0.7 to 4.5 ± 0.7 and did not differ among themselves (P > 0.05). Piglet birth weight was greater (P < 0.05) for sows supplied with 28 g/d microalgae than with lower levels (Table 2). Among the the three tested covariates, total litter size and sex of the piglets had significant effects on piglet birth weight (P < 0.05), whereas parity did not present a significant effect (P > 0.05). Total and born alive litter sizes at the subsequent parity were 14.7 ± 3.2 and 13.5 ± 3.0, respectively. Total litter size and stillborn rates did not differ (P > 0.05) for sows receiving distinct levels of supplementation (Table 3). Total litter size for parity 3-4 sows (15.0 ± 0.3) was greater (P < 0.05) than for sows with 5 (14.8 ± 0.4) and 6-7 parities (13.8 ± 0.3). Stillborn rates ranged from 4.3 ± 0.6 to 4.9 ± 0.7 for sows with 3, 4 and 5 parities, and were lower (P < 0.05) than for sows with 6-7 parities (7.0 ± 0.5). Serum cholesterol levels during gestation (57.7 ± 1.9 mg/dl) were lower (P < 0.05) than during lactation and the WEI (81.2 ± 1.9 and 81.1 ± 1.9 mg/dl, respectively). Likewise, serum levels of IGF-1 during lactation (240.6 ± 11.7 mg/dl) and the WEI (224.0 ± 11.7) were greater (P < 0.05) than during gestation (37.6 ± 11.7 mg/dl). In contrast, serum levels of NEFA were greater (P < 0.05) during gestation (918.6 ± 39.6 μmol/l) than during lactation (587.2 ± 38.9 μmol/l) and the WEI (676.9± 38.9 μmol/l). Nonetheless, serum levels of cholesterol, IGF-1 and NEFA did not differ among treatments (P > 0.05). Due to a treatment per period interaction (Figure 1), serum levels of triglycerides were inferior for sows supplemented with 14.0 and 28.0 g/d microalgae than for sows in other treatments, only during gestation (P < 0.05), but no other differences occurred (P > 0.05). No effect of parity was observed on any metabolic parameter (P > 0.05).
4. Discussion This was the first study testing supplementation with DHA from heterotrophic microalgae in sow diets. Although that supplementation did not improve WEI at the parity concurrent to supplementation, total litter size at the subsequent parity and stillborn rates at either parity, piglet birth weight was greater for sows fed 28.g/d than for sows fed lower levels. The improvement on piglet birth weight observed in the present study may be attributed to positive effects on uterine environment that favor embryo survival and viability (Smits et al. 2011). It is expected that piglet birth weight would be reduced in greater litters and that male piglets would be heavier than females, as confirmed by the tested significant covariates. However, as previous analyses indicated that none of such covariates presented interactions with the main response of interest, the effect of the supplementation with PUFA on piglet birth weight cannot be attributed to either sex or litter size. Improved piglet birth weight following supplementation with PUFA has also been reported in some studies (Corino et al. 2009; Mateo et al. 2014), although such an effect eventually may not be evident (Smit et al 2013). Our findings sustain the controversies surrounding supplementation with PUFA. Reduction in the WEI after supplementation with fish oil was not observed (Smits et al. 2011; Mateo et al. 2014), even with intervals longer than those observed in the present study. Following supplementation with PUFA from other sources, detrimental effects on total litter size (Smit et al. 2015) and no benefit for stillborn rates (Mateo et al. 2014) were reported, whereas improved stillborn rates were reported elsewhere after supplementation with fish oil (Tanghe et al. 2014). Increased litter size was reported for sows supplemented with fish oil (Smits et al. 2011), but with a mean much lower (10.7) than that observed in the present study (14.0). Nevertheless, eventual benefits on litter size and birth weight may result in prolonged farrowings (Tanghe and de Smet 2013)
and on increased stillborn rates (Lucia et al. 2002). In the present study, the low stillborn rate may reflect the fact that many farrowings were supervised at the farm where the study was conducted, which also presented total litter size and WEI at excellent levels according to industry benchmarks (Agriness 2016). Our results suggest that supplementation with omega-3 PUFA from microalgae may not be beneficial with high levels of reproductive performance. Serum levels of triglycerides were decreased during gestation for sows fed 14.0 and 28.0 g/d microalgae, even though sows fed lower concentrations presented during gestation levels as high as those observed during lactation and after weaning. A decrease in circulating levels of triglycerides normally occurs throughout the last third part of the gestation, due to the combined effect of the accelerated piglets’ growth with the increased function of the mammary glands (Mosnier et al. 2010). Reduced levels of triglycerides were also observed after supplementation of female rats with PUFA, in both mothers and puppies (Lombardi et al. 2001), due to decrease in lipogenesis at the liver, particularly because of inhibition of the secretion of low-density lipoproteins (Harris and Bulchandani 2006). Yet, the mechanisms explaining such effect in sow’s metabolism are still to the determined. Generally, the other the tested metabolic markers were unaffected by distinct levels of supplementation, as reported for IGF-1 and NEFA after supplementation with linoleic acid (Corino et al. 2009) and for IGF-1 and triglycerides for pre-pubertal gilts supplemented with fish oil (Moreira et al. 2016). However, reduced serum cholesterol levels were observed for pre-pubertal gilts supplemented with fish oil (Adkins and Kelley 2010; Moreira et al. 2016). During lactation, when sows commonly face negative energy balance, increased circulatory levels of NEFA would be expected, since NEFA is an indicator of fat mobilization (Prunier and Quesnel 2000). However, in the
present study, serum concentration of NEFA were reduced during lactation, which may have resulted from the high content of unsaturated lipids present in the microalgae (Ryckebosch et al. 2012; Martins et al. 2013). That would favor the establishment of energy reserves during lactation, as a metabolic adaption developed by the supplemented sows to keep their milk production (Prunier and Quesnel 2000). Under negative energy balance, serum levels of insulin and IGF-1 may not be enough to sustain normal folliculogenesis (Quesnel et al. 2009). Thus, increased expenditure of energy reserves resulting from the supplementation with PUFA may lead to increased concentration of both such metabolites, contributing to improve the quality of ovarian follicles, which should be investigated by further research. Heterotrophic microalgae present greater DHA content than other sources of PUFA (Ryckebosch et al. 2012; Martins et al. 2013), which might alter organoleptic traits of the diet. However, our research team did not notice any unpleasant smell in the diets or any sows rejecting diets during the experiment. Also, the amount of supplement added to the diets, as recommended by the manufacturer, was very small considering the total volume of the diets. Future research in that field may consider adjusting the DHA content according to the omega-6:omega-3 ratio, as done in a study that reported benefits for the sperm quality of boars supplemented with fish oil (Liu et al. 2016). 5. Conclusions Supplementation of sow diets with heterotrophic microalgae Schizochytium sp. had no influence on stillborn rates, on the weaning-estrous interval and on total litter size. For sows fed the greatest tested concentration (28.0 g/d), serum levels of triglycerides were reduced during gestation and piglet birth weights were improved.
Funding
This research was funded with a scholarship given to the first author by CAPES and with a research grant given to T Lucia Jr. by CNPq.
Conflict of interest The authors have no conflict of interest to declare.
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Fig. 1. Serum levels of triglycerides in sows supplemented with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during three periods: last 30 days of gestation; lactation (21.9 ± 0.9 days); and the WEI (3.5 ± 0.8 days)* *Means ± SEM differ according to the treatment by period interaction (P < 0.05)
Table 1. Stillbirth rate and weaning-estrous interval (WEI) at the parity concurrent to the supplementation of sows with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during the last 30 days of gestation, lactation (21.9 ± 0.9 days) and the WEI (3.5 ± 0.8 days) Level (g/d)
Stillbirths/farrowing*
Weaning-estrous interval*
n
(%)
n
Days
0
121
3.3 ± 0.8
89
3.3 ± 0.1
3.5
123
5.0 ± 0.8
106
3.6 ± 0.1
7.0
114
4.7 ± 0.8
88
3.7 ± 0.1
14.0
123
5.2 ± 0.8
96
3.4 ± 0.1
ab
28.0
115
4.3 ± 0.8
85
3.5 ± 0.1
ab
Total
596
4.5 ± 0.5
463
a,b
a
ab
b
3.2 ± 0.1
Means ± SEM with distinct superscripts differ in the column by at least P < 0.05
Table 2. Piglet birth weight at the parity concurrent to the supplementation of sows with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during the last 30 days of gestation, lactation (21.9 ± 0.9 days) and the WEI (3.5 ± 0.8 days) Level (g/d)
Farrowings (n)
Piglets (n)
Piglet birth weight (kg)
0
10
129
1.26 ± 0.03b
3.5
12
153
1.26 ± 0.03b
7.0
11
169
1.29 ± 0.02b
14.0
12
175
1.29 ± 0.02b
28.0
10
146
1.41 ± 0.03a
Total
55
772
1.30 ± 0.01
a,b
Means ± SEM with distinct superscripts differ in the column by at least P < 0.05
Table 3. Total litter size and stillbirth rate at the parity subsequent to the supplementation of sows with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during the last 30 days of gestation, lactation (21.9 ± 0.9 days) and the weaning-estrous interval (3.5 ± 0.8 days). Level (g/d)
n
Total litter size
Stillbirths/farrowing(%)
0
88
14.7 ± 0.4
5.0 ± 0.7
3.5
106
14.6 ± 0.3
4.6 ± 0.6
7.0
88
14.2 ± 0.4
5.5 ± 0.7
14.0
96
14.9 ± 0.3
6.5 ± 0.7
28.0
85
14.8 ± 0.4
4.6 ± 0.7
Total
463
14.7 ± 0.3
5.4 ± 0.5
*
Means ± SEM did not differ (P > 0.05)
Highlights
Sow diets were supplemented with five levels of omega-3 from microalgae
Supplementation lasted from the 85th gestation day until the weaning-to-estrous interval
There was no effect on stillbirths at both the concurrent and the subsequent parity
Total litter size at the subsequent parity was not increased
Piglet birth weight was improved at the highest level of supplementation
Lower serum levels of triglycerides occurred during gestation for sows fed 14.0 and 28.0 g/d.
PII: DOI: Reference:
S1871-1413(17)30333-5 https://doi.org/10.1016/j.livsci.2017.11.006 LIVSCI3344
To appear in: Livestock Science Received date: 7 March 2017 Revised date: 25 October 2017 Accepted date: 2 November 2017 Cite this article as: C.J.M. Posser, L.M. Almeida, F. Moreira, I. Bianchi, B.G. Gasperin and T. Lucia, Supplementation of diets with omega-3 fatty acids from microalgae: effects on sow reproductive performance and metabolic parameters, Livestock Science, https://doi.org/10.1016/j.livsci.2017.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supplementation of diets with omega-3 fatty acids from microalgae: effects on sow reproductive performance and metabolic parameters C.J.M. Posser1, L.M. Almeida2, F. Moreira1, I. Bianchi3, B.G. Gasperin1, T. Lucia Jr. 1* 1
ReproPel, Faculdade de Veterinária, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil 2
Faculdade Evangélica do Paraná, 80730-000, Curitiba-PR, Brazil 3
Instituto Federal Catarinense, 89245-000, Araquari-SC, Brazil
Abstract Supplementation of diets with omega-3 polyunsaturated fatty acids (PUFA) may benefit sow reproductive performance, but the efficiency of algae as a source of PUFA is still unknown. This study evaluated serum levels of metabolic markers and reproductive performance of sows supplemented with the heterotrophic microalgae Schizochytium sp. Sows (n = 596) were supplemented from the 85th day of gestation, during lactation and until the weaning-estrous interval (WEI). Microalgae were included at five levels: 0 (control); 3.5; 7.0; 14.0; and 28.0 g/d. Blood samples were collected at the 85th day of gestation, at the 10th d of lactation and at the last day prior to weaning (WEI). Serum levels of cholesterol, triglycerides, insulin-like growth factor type-1 (IGF-1) and nonesterified fatty acids (NEFA) were evaluated. At the parity concurrent with the supplementation, no effects were observed on stillborn rates (P > 0.05), sows fed 28.0 g/d farrowed heavier piglets than sows fed lower levels (P < 0.05), but the WEI for sows fed 7.0 g/d was longer than for the control (P < 0.05). Serum levels of triglycerides were decreased during gestation, only for sows supplemented with 14.0 and 28.0 g/d (P < 0.05). At the subsequent parity, there was no difference on stillborn rates and total Author’s address (for correspondence): T. Lucia Jr., ReproPel, Faculdade de
*
Veterinária, Universidade Federal de Pelotas, Pelotas, RS 96010-900, Brazil. E-mail: [email protected]
litter size (P > 0.05). The highest tested concentration (28.0 g/d) of Schizochytium sp. was associated with reduced serum levels of triglycerides during gestation and with increased piglet birth weight, but the supplementation generally did not benefit sow reproductive performance. Key words: DHA, litter size, stillbirths, birth weight, metabolism, sows.
1. Introduction Total litter size has increased over time in swine due to genetic selection for prolifacy, concurrently with an increase in stillborn rates and in the number of piglets born with low viability (reviewed by Prunier et al. 2010). Thus, strategies to improve post-natal piglet development are of interest to the swine industry. Gestation and lactation diets may be supplemented with polyunsaturated fatty acids (PUFA), especially those of the omega-3 series (reviewed by Rossi et al. 2010). Omega-3 PUFA can be incorporated into oocytes, as observed for cattle and sheep (Zachut et al. 2008; Gulliver et al. 2012) and into tissues of swine embryos and fetuses (Brazle et al. 2009), potentially benefiting their subsequent development. Omega-3 PUFA may also affect metabolic markers, such as triglycerides, non-esterified fatty acids (NEFA), insulin-like growth factor type 1 (IGF-1) and cholesterol, influencing energy metabolism and the function of the reproductive axis (Prunier and Quesnel 2000; Harris and Bulchandani 2006; Adkins and Kelley 2010). Despite of reported benefits on fetal development (Innis 1991) and on litter size (Smits et al. 2011), effects on fetal survival may be inconsistent (Perez Rigau et al. 1995). Thus, the effects of supplementation with omega-3 PUFA on sow performance are still controversial, especially due the variety of their available sources.
The docosahexaenoic acid (DHA) is one of the most important omega-3 PUFA (reviewed by Rossi et al. 2010; and by Tanghe and de Smet 2013). The DHA may have several sources, including vegetable (Ryckebosch et al. 2012), fish (Moreira et al. 2016) and marine oils (Smit et al. 2013; 2015). Fish oils are the most commonly used sources, but their use may be questioned due: low availability; inconsistent nutritional profile; risk of contamination by chemicals or pollutants deposited at the sea; and sustainability issues (Ryckebosch et al. 2012). On the other hand, omega-3 PUFA extracted from algae are non-pathogenic and nontoxigenic (Martins et al. 2013), representing a lowcost sustainable source with stable nutritional levels, high lipid content and greater DHA content than vegetable oils (Ryckebosch et al. 2012; Martins et al. 2013). Nevertheless, effects of supplementation with PUFA from algae on metabolism and reproductive performance of sows are not yet known. The objective of the present study was to evaluate the effects of supplementing gestation and lactation diets with heterotrophic microalgae on sow’s reproductive performance and metabolic parameters.
2. Materials and methods 2.1. Experimental design The study was conducted in a commercial farm with an inventory of 3,400 F1 crossbred females (PIC Camborough®) located in southern Brazil (latitude 27°22’35”S, longitude 51°02’38”W). All experimental procedures were approved by the Ethics in Animal Experimentation Committee of the Universidade Federal de Pelotas. The study was designed to include 650 sows. In each weekly group, sows were randomly assigned to five treatments (n = 130 sows each). Such sows had no locomotion problems and were neither used as nurses during lactation, nor previously programmed to be culled after lactation. Thereafter, 54 sows were censored: those that had unplanned culling
during the experiment; and those with unreliable records for dates of WEI and artificial inseminations. Sows were not paired by parity within treatments, due to logistic constraints of the farm’s production flow. The parity distribution of the remaining 596 sows was as follows: 2 (n = 149); 3 (n = 128); 4 (n = 104); 5 (n = 94); and 6-7 parities (n = 121). A commercial source of heterotrophic microalgae Schizochytium sp. (All-GRich, Alltech Inc.) containing 120 g/kg DHA (Supplementary material) was added to the diets, during the last 30 d of gestation and the lactation (21.9 ± 0.9 d), until the weaning-estrous interval (WEI) (3.5 ± 0.8 d). The supplement was a seaweed meal which was top-dressed to the diet on an individual basis, to guarantee that all sows were fed the same amount of the product. The treatments represented five supplementation levels: 0 (control, n = 121); 3.5 (n = 123); 7.0 (n = 114); 14.0 (n = 123); and 28.0 g/d (n = 115). During gestation, sows were kept in individual crates with automatic drop feeders, receiving 2-3 kg/d of a gestation diet containing: 14.4% crude protein; 3,100 Kcal/kg ME; and 3.2% fat. The amount of the diet was adjusted to each sow’s body condition. Prior to the predicted farrowing date (2-3 d), sows were housed at farrowing rooms, with ad libitum access to feed after the 4th d of lactation. The lactation diet contained: 20.1% crude protein; 3,337 Kcal/kg ME; and 4.8% fat. After weaning, sows were kept in individual crates and submitted to flushing with the lactation diet, until detected in estrous. Estrous detection was done conducted once a day, through back pressure in the presence of a sexually mature boar. Artificial insemination was done at 24 h-intervals, via intracervical, with 3 x 109 viable spermatozoa (2.4 AI per sow, on average).
2.2. Metabolic analyses From 45 sows selected at random within the same weekly weaning group (n = 9 per treatment), blood samples were collected from the jugular vein at: the 85th d of gestation; the 10th d of lactation; and the last d before weaning. After centrifugation at 7,000 x g for 5 min, the serum was placed inside cryotubes and stored in liquid nitrogen. Serum levels of cholesterol (11539, BioSystems, Curitiba, PR, Brazil; sensitivity 0.3 mg/dl) and triglycerides (87.1/250, Labtest, Lagoa Santa, MG, Brazil; sensitivity 3.0 mg/dl) were evaluated through standard enzymatic procedures. Chemiluminescence was used to assay free IGF-1, after acid treatment to allow precise quantification (Siemens Immulite 2000 IGF-1 L2KGF2: Siemens Healthcare Ltda, São Paulo, SP, Brazil; sensitivity 20.0 ng/mL). Serum levels of NEFA were evaluated by spectrophotometry (NEFA-HR2 Wako, Neuss, Germany; sensitivity 1.0 mEq/L). All assays were conducted in duplicate, in a commercial laboratory, using kits developed for human serum, but with multi-species validation (all CV inferior to 10%). 2.3. Reproductive performance At the parity concurrent to the supplementation, the number of total pigs born and stillborn was recorded at farrowing and the WEI was recorded. Stillborn rates per farrowing were calculated by dividing the number of stillborns in each litter by the total litter size, to adjust such rate for total litter size. For 55 randomly selected farrowings, individual piglet birth weights were determined using a computerized scale (Urano® US 20/2 pop light). The sex of the weighted piglets was also recorded. At the subsequent parity, total litter size and stillborn rates per farrowing were also recorded.
2.4. Statistical analyses Total litter size, stillborn rates and WEI were compared among treatments and parities through analyses of variance. As stillborn rates and WEI were not normally distributed, such variables were submitted to an angular transformation (arcsine of the square root of each value/100). Piglet birth weight at the parity concurrent to the supplementation was compared among treatments, with adjustment for the following covariates: parity of the sow; total litter size; and sex of each piglet. Serum levels of cholesterol, triglycerides, IGF-1 and NEFA were compared across treatments and periods of sample collection by analyses of variance with repeated measures, considering parity as a random factor and the individual effect of sows nested within the effect of period. Due to lack of normality, serum cholesterol levels were transformed to the logarithmic scale. In all models, comparisons of means were conducted using Tukey test. All analyses were conducted using Statistix® (2014).
3. Results No significant treatment per parity interaction affected the WEI at the parity concurrent with the supplementation and stillborn rates at both tested parities (P > 0.05). The WEI for sows supplemented with 7.0 g/d microalgae was longer (P < 0.05) than for control group (Table 1). Across parities, WEI was longer (P < 0.05) for sows with 2 (3.6 ± 0.1) and 6-7 parities (3.7 ± 0.1) than for sows with 3 and 4 parities (3.4 ± 0.1 and 3.3 ± 0.1, respectively). At the parity concurrent with the supplementation, total litter size was 14.4 ± 3.5, and born alive litter size was 13.4 ± 3.4. The percent of stillbirths was not influenced (P > 0.05) by the treatments (Table 1). Stillborn rates for sows with 6-7
parities (6.6 ± 0.7) were greater than at lower parities (P < 0.05), which ranged from 4.2 ± 0.7 to 4.5 ± 0.7 and did not differ among themselves (P > 0.05). Piglet birth weight was greater (P < 0.05) for sows supplied with 28 g/d microalgae than with lower levels (Table 2). Among the the three tested covariates, total litter size and sex of the piglets had significant effects on piglet birth weight (P < 0.05), whereas parity did not present a significant effect (P > 0.05). Total and born alive litter sizes at the subsequent parity were 14.7 ± 3.2 and 13.5 ± 3.0, respectively. Total litter size and stillborn rates did not differ (P > 0.05) for sows receiving distinct levels of supplementation (Table 3). Total litter size for parity 3-4 sows (15.0 ± 0.3) was greater (P < 0.05) than for sows with 5 (14.8 ± 0.4) and 6-7 parities (13.8 ± 0.3). Stillborn rates ranged from 4.3 ± 0.6 to 4.9 ± 0.7 for sows with 3, 4 and 5 parities, and were lower (P < 0.05) than for sows with 6-7 parities (7.0 ± 0.5). Serum cholesterol levels during gestation (57.7 ± 1.9 mg/dl) were lower (P < 0.05) than during lactation and the WEI (81.2 ± 1.9 and 81.1 ± 1.9 mg/dl, respectively). Likewise, serum levels of IGF-1 during lactation (240.6 ± 11.7 mg/dl) and the WEI (224.0 ± 11.7) were greater (P < 0.05) than during gestation (37.6 ± 11.7 mg/dl). In contrast, serum levels of NEFA were greater (P < 0.05) during gestation (918.6 ± 39.6 μmol/l) than during lactation (587.2 ± 38.9 μmol/l) and the WEI (676.9± 38.9 μmol/l). Nonetheless, serum levels of cholesterol, IGF-1 and NEFA did not differ among treatments (P > 0.05). Due to a treatment per period interaction (Figure 1), serum levels of triglycerides were inferior for sows supplemented with 14.0 and 28.0 g/d microalgae than for sows in other treatments, only during gestation (P < 0.05), but no other differences occurred (P > 0.05). No effect of parity was observed on any metabolic parameter (P > 0.05).
4. Discussion This was the first study testing supplementation with DHA from heterotrophic microalgae in sow diets. Although that supplementation did not improve WEI at the parity concurrent to supplementation, total litter size at the subsequent parity and stillborn rates at either parity, piglet birth weight was greater for sows fed 28.g/d than for sows fed lower levels. The improvement on piglet birth weight observed in the present study may be attributed to positive effects on uterine environment that favor embryo survival and viability (Smits et al. 2011). It is expected that piglet birth weight would be reduced in greater litters and that male piglets would be heavier than females, as confirmed by the tested significant covariates. However, as previous analyses indicated that none of such covariates presented interactions with the main response of interest, the effect of the supplementation with PUFA on piglet birth weight cannot be attributed to either sex or litter size. Improved piglet birth weight following supplementation with PUFA has also been reported in some studies (Corino et al. 2009; Mateo et al. 2014), although such an effect eventually may not be evident (Smit et al 2013). Our findings sustain the controversies surrounding supplementation with PUFA. Reduction in the WEI after supplementation with fish oil was not observed (Smits et al. 2011; Mateo et al. 2014), even with intervals longer than those observed in the present study. Following supplementation with PUFA from other sources, detrimental effects on total litter size (Smit et al. 2015) and no benefit for stillborn rates (Mateo et al. 2014) were reported, whereas improved stillborn rates were reported elsewhere after supplementation with fish oil (Tanghe et al. 2014). Increased litter size was reported for sows supplemented with fish oil (Smits et al. 2011), but with a mean much lower (10.7) than that observed in the present study (14.0). Nevertheless, eventual benefits on litter size and birth weight may result in prolonged farrowings (Tanghe and de Smet 2013)
and on increased stillborn rates (Lucia et al. 2002). In the present study, the low stillborn rate may reflect the fact that many farrowings were supervised at the farm where the study was conducted, which also presented total litter size and WEI at excellent levels according to industry benchmarks (Agriness 2016). Our results suggest that supplementation with omega-3 PUFA from microalgae may not be beneficial with high levels of reproductive performance. Serum levels of triglycerides were decreased during gestation for sows fed 14.0 and 28.0 g/d microalgae, even though sows fed lower concentrations presented during gestation levels as high as those observed during lactation and after weaning. A decrease in circulating levels of triglycerides normally occurs throughout the last third part of the gestation, due to the combined effect of the accelerated piglets’ growth with the increased function of the mammary glands (Mosnier et al. 2010). Reduced levels of triglycerides were also observed after supplementation of female rats with PUFA, in both mothers and puppies (Lombardi et al. 2001), due to decrease in lipogenesis at the liver, particularly because of inhibition of the secretion of low-density lipoproteins (Harris and Bulchandani 2006). Yet, the mechanisms explaining such effect in sow’s metabolism are still to the determined. Generally, the other the tested metabolic markers were unaffected by distinct levels of supplementation, as reported for IGF-1 and NEFA after supplementation with linoleic acid (Corino et al. 2009) and for IGF-1 and triglycerides for pre-pubertal gilts supplemented with fish oil (Moreira et al. 2016). However, reduced serum cholesterol levels were observed for pre-pubertal gilts supplemented with fish oil (Adkins and Kelley 2010; Moreira et al. 2016). During lactation, when sows commonly face negative energy balance, increased circulatory levels of NEFA would be expected, since NEFA is an indicator of fat mobilization (Prunier and Quesnel 2000). However, in the
present study, serum concentration of NEFA were reduced during lactation, which may have resulted from the high content of unsaturated lipids present in the microalgae (Ryckebosch et al. 2012; Martins et al. 2013). That would favor the establishment of energy reserves during lactation, as a metabolic adaption developed by the supplemented sows to keep their milk production (Prunier and Quesnel 2000). Under negative energy balance, serum levels of insulin and IGF-1 may not be enough to sustain normal folliculogenesis (Quesnel et al. 2009). Thus, increased expenditure of energy reserves resulting from the supplementation with PUFA may lead to increased concentration of both such metabolites, contributing to improve the quality of ovarian follicles, which should be investigated by further research. Heterotrophic microalgae present greater DHA content than other sources of PUFA (Ryckebosch et al. 2012; Martins et al. 2013), which might alter organoleptic traits of the diet. However, our research team did not notice any unpleasant smell in the diets or any sows rejecting diets during the experiment. Also, the amount of supplement added to the diets, as recommended by the manufacturer, was very small considering the total volume of the diets. Future research in that field may consider adjusting the DHA content according to the omega-6:omega-3 ratio, as done in a study that reported benefits for the sperm quality of boars supplemented with fish oil (Liu et al. 2016). 5. Conclusions Supplementation of sow diets with heterotrophic microalgae Schizochytium sp. had no influence on stillborn rates, on the weaning-estrous interval and on total litter size. For sows fed the greatest tested concentration (28.0 g/d), serum levels of triglycerides were reduced during gestation and piglet birth weights were improved.
Funding
This research was funded with a scholarship given to the first author by CAPES and with a research grant given to T Lucia Jr. by CNPq.
Conflict of interest The authors have no conflict of interest to declare.
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Fig. 1. Serum levels of triglycerides in sows supplemented with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during three periods: last 30 days of gestation; lactation (21.9 ± 0.9 days); and the WEI (3.5 ± 0.8 days)* *Means ± SEM differ according to the treatment by period interaction (P < 0.05)
Table 1. Stillbirth rate and weaning-estrous interval (WEI) at the parity concurrent to the supplementation of sows with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during the last 30 days of gestation, lactation (21.9 ± 0.9 days) and the WEI (3.5 ± 0.8 days) Level (g/d)
Stillbirths/farrowing*
Weaning-estrous interval*
n
(%)
n
Days
0
121
3.3 ± 0.8
89
3.3 ± 0.1
3.5
123
5.0 ± 0.8
106
3.6 ± 0.1
7.0
114
4.7 ± 0.8
88
3.7 ± 0.1
14.0
123
5.2 ± 0.8
96
3.4 ± 0.1
ab
28.0
115
4.3 ± 0.8
85
3.5 ± 0.1
ab
Total
596
4.5 ± 0.5
463
a,b
a
ab
b
3.2 ± 0.1
Means ± SEM with distinct superscripts differ in the column by at least P < 0.05
Table 2. Piglet birth weight at the parity concurrent to the supplementation of sows with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during the last 30 days of gestation, lactation (21.9 ± 0.9 days) and the WEI (3.5 ± 0.8 days) Level (g/d)
Farrowings (n)
Piglets (n)
Piglet birth weight (kg)
0
10
129
1.26 ± 0.03b
3.5
12
153
1.26 ± 0.03b
7.0
11
169
1.29 ± 0.02b
14.0
12
175
1.29 ± 0.02b
28.0
10
146
1.41 ± 0.03a
Total
55
772
1.30 ± 0.01
a,b
Means ± SEM with distinct superscripts differ in the column by at least P < 0.05
Table 3. Total litter size and stillbirth rate at the parity subsequent to the supplementation of sows with distinct levels of heterotrophic microalgae Schizochytium sp., containing 120 g/kg docosahexaenoic acid, during the last 30 days of gestation, lactation (21.9 ± 0.9 days) and the weaning-estrous interval (3.5 ± 0.8 days). Level (g/d)
n
Total litter size
Stillbirths/farrowing(%)
0
88
14.7 ± 0.4
5.0 ± 0.7
3.5
106
14.6 ± 0.3
4.6 ± 0.6
7.0
88
14.2 ± 0.4
5.5 ± 0.7
14.0
96
14.9 ± 0.3
6.5 ± 0.7
28.0
85
14.8 ± 0.4
4.6 ± 0.7
Total
463
14.7 ± 0.3
5.4 ± 0.5
*
Means ± SEM did not differ (P > 0.05)
Highlights
Sow diets were supplemented with five levels of omega-3 from microalgae
Supplementation lasted from the 85th gestation day until the weaning-to-estrous interval
There was no effect on stillbirths at both the concurrent and the subsequent parity
Total litter size at the subsequent parity was not increased
Piglet birth weight was improved at the highest level of supplementation
Lower serum levels of triglycerides occurred during gestation for sows fed 14.0 and 28.0 g/d.