In comparison to many other species, cryopreservation of stallion sperm results in significant cellular damage which compromises sperm function in artificial reproduction efforts. It has been hypothesized that sperm membrane fragility may be due to the higher content of docosapentanoic acid (DPA; 22:5) relative to docosahexaenoic acid (DHA; 22:6) in stallion sperm. This ratioin stallion sperm appears to be the reverse of that in sperm of most other livestock species. Prvious studies have demonstrated that DHA supplementation can improve the viability of equine sperm, however, the structural lipids involved have not been examined. We therefore undertook adietary supplementation study to determine if augmented DHA levels results in increased levels of sperm glycerophospholipids containing DHA. A targeted lipidomics analysis of DHA-containing glycerophospholipids was conducted for spermatozoa obtained from stallions prior to and upon completion of 60 days of dietary yeast-based supplement containing algal DHA. Control stallions were fed a comparable diet without supplementation.These analyses demonstrate for the first time that increasing free DHA levels in spermatozoaresults in utilization of the DHA in lipid remodeling to augment levels of DHA-containing choline and ethanolamine plasmalogens and diacyl glycerophospholipids in spermatozoa. We have generated the first detailed lipidomics data of the effects of supplementation in stallions, providing the database required for evaluating the hypothesis that low DHA levels are responsible for the poor viability of cryopreserved equine sperm.

•    Stallion
•    Spermatozoa
•    Glycerophospholipids
•    Docosahexaenoic acid

Wood PL, Scoggin K, Ball BA, Lawrence L, Troedsson MH, et al. (2017) Lipidomics Evaluation of the Effects of Feeding a Yeast-Based Supplement Containing Algal Docosahexaenoic Acid (DHA) on DHA-Containing Glycerophospholipids in Stallion Spermatozoa. J Vet Med Res 4(2): 1078.

Reproductive technologies in the equine industry, particularly as it relates to cryopreservation, are essential for international trade and efficient use of male germplasm [1]. Stallion sperm do not survive the freezing and thawing process as well as other species such as the bull. It has been suggested that this lowered freezability of stallion sperm may be due the higher content of docosapentanoic acid (DPA; 22:5) relative to docosahexaenoic acid (DHA; 22:6), a ratio that is the reverse for these polyunsaturated fatty acids (PUFAs) in most other livestock species [2-6]. Because there is limited information demonstrating that DHA supplementation augments DHAcontaining glycerophospholipids in stallion spermatozoa we undertook a targeted evaluation of the equine sperm lipidome pre- and post supplementation. These glycerophospholipids are thought to be critical structural lipids that are essential for male fertility due to their roles in membrane fluidity and fusogenicity [3-5]. In particular, recent studies indicate that differences in fatty acid and plasmalogen composition of stallion spermatozoa are associated with differences in survival of sperm after cryopreservation as well as susceptibility to lipid peroxidation [3-6 membrane and results in an improvement of sperm motility after cooling to 5o C or after freezing/thawing [7-11].While these studies evaluated the effects of DHA supplementation on total glycerophospholipid pools, individual lipids were not evaluated.

Since PUFAs provide fluidity and flexibility to membranes these structural alterations are thought to minimize cold shock damage. Additionally, since DHA supplementation has been reported to enhance sperm viability in stallions [7] and other species [8-11], increasing our knowledge of the effects of DHA supplementation on individual lipids in the equine sperm lipidome is essential to advance our ability to develop techniques to improve the stability and viability of stallion spermatozoa. To examine this question, we undertook a high-resolution mass spectrometric targeted lipidomics analysis of stallion spermatozoa pre- and post-60 days of supplementation with or without DHAfrom an algal source.

Semen collection and processing (IACUC approved)

Stallions (n = 12) had semen collected with an artificial vagina on an alternate-day basis for six collections to stabilize extragonadal sperm reserves. Stallions were randomly assigned to either the control or supplementation group. Control stallions(N=6) were fed a basal diet of mixed grass hay fed at 1.8% BW and a concentrate fed at 0.4% BW for 60 days to meet the nutritional requirements for mature, idle horses (NRC). In the treatment group, stallions (N=6) received the same diet as the control group plus 60 g of a yeast-based supplement containing 2 mg selenium as selenized yeast, 1000 IU vitamin E and a 15 g algal DHA (Alltech Inc., Nicholasville, KY) for 60 days.

All horses received the basal diet during the pre-treatment collection period (12 days). Stallions were housed in individual box stalls and had access to a turnout pen every other day.

Beginning at 50 days after the initiation of supplementation, stallions again had semen collected on an alternate-day basis for six collections. For both the pretreatment and post-treatment period, semen collected at the sixth collection was processed for evaluation of sperm lipidomics. For separation of intact sperm from seminal plasma, semen was diluted (1:1) with cold PBS and centrifuged at 800xg for 20 minutes. The supernatant was removed leaving only the intact sperm pellet. The resulting pellet was resuspended in 10 mL of cold PBS and centrifuged a second time (800xg; 20 min) prior to removal of the supernatant and resupension of the pellet in 1.0 mL of PBS which was snap frozen in liquid nitrogen for storage prior to analysis. This procedure does not remove all seminal plasma components from washed spermatozoa since some seminal plasma components are adherent to ejaculated sperm.

Spermatozoa lipidomics

After thawing, 100 µL of sperm suspension (1 – 2 mg of protein) were sonicated in 1 mL of water and 1 mL of methanol containing [2 H8 ]arachidonic acid, [2 H4 ]hexacosanoic acid, [13C18] stearic acid,[2 H31]PtdE 34:1, [2 H54]PtdE 28:0, [2 H31]PtdC 34:1, [2 H54]PtdC 28:0, [2 H62]PtdC 32:0, [2 H31]SM 16:0, [2 H31]PS 36:1, [2 H31]PA 34:1, [2 H62]PG 32:0, and bromocriptine as internal standards [6]. Next 2 mL of methy-tert-butyl ether were added and the tubes were vigorously shaken at room temperature for 30 min. Organic extracts were dried by centrifugal vacuum evaporation and dissolved in isopropanol: methanol: chloroform (4:2:1) containing 15 mM ammonium acetate.

Direct infusion lipidomics were performed utilizing highresolution (140,000 at 200 amu; 0.2 to 3 ppm mass error) data acquisition, on an orbitrap mass spectrometer (Thermo Q Exactive) with successive switching between polarity modes [6,12-15]. In negative ion ESI, the anions of ethanolamine plasmalogens (PlsE), phosphatidylethanolamines (PtdE), lysophosphoethanolamines (LPE), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidylserines (PS), and fatty acids were quantitated and lipid identities validated by MS/ MS. In positive ion ESI, the cations of choline plasmalogens (PlsC) and phosphatidylcholines (PtdC), were quantitated and lipid identities validated by MS/MS. The cations and anions of bromocriptine were used to monitor for potential mass axis drift. Between injections, the transfer line was washed with successive 500 µL washes of methanol and hexane/ethyl acetate (3:2) to minimize ghost effects.

Protein determination

Proteins were determined utilizing the Pierce BCA Protein Assay kit (Thermo Fisher).

Statistical analysis

R values (ratio of endogenous lipid peak area to the peak area of an appropriate internal standard) were calculated and corrected for protein. Data are presented as mean ± SEM. A paired t-test was used to determine significant differences (p < 0.05) between pre- and post-supplementation in the experimental group and between pre- and post-control diet in the control group.

Spermatozoal free fatty acids

The major polyunsaturated free fatty acids monitored in equine sperm were 22:5 (DPA, docosapentaenoic acid) and 22:6 (DHA; docosahexaenoic acid). Free DHA levels were augmented by supplementation resulting in a significantly elevated DHA/ DPA ratio in sperm (Table 1).

Spermatozoal choline diacyl and alkenyl-acyl glycerophospholipids

High levels of diacyl (phosphatidylcholines, PtdC) and alkenylacyl (choline plasmalogens, PlsC) glycerophosphocholines, containing polyunsaturated fatty acids at sn-2 of the glycerol backbone, were measured in stallion sperm. Members of these lipid families with DHA at sn-2 were augmented by dietary DHA whilePtdC 36:5 had decreased incorporation of 22:5 at sn-2 (Figure 1). MS2 of these lipids revealed the characteristic phosphocholine fragment (184.0738) but did not allow us to verify the fatty acid substitutions. No pre- vs. post-differences in these lipids were monitored in the control group.

Spermatozoal ethanolamine diacyl and alkenyl-acyl glycerophospholipids

Similar to choline glycerophospholipids, supplementation resulted in augmented levels of DHA-containing ethanolamine glycerophospholipids. This included phosphatidylethanolamines (PtdE) and ethanolamine plasmalogens (PlsE) as presented in Figure (2). The identities of the PtdEs were validated by MS2 for both the sn-1 and sn-2 fatty acid substituents and the sn-2 fatty acid substituents of PlsEs (Table 2). No pre- vs. post-differences in these lipids were monitored in the control group.

Spermatozoal serine diacyl glycerophospholipids

Supplementation also resulted in augmented levels of the DHA-containing phosphatidylserine (PtdS), PtdS 40:6 (Figure 2). The identities of PtdS 40:5 and 40:6 were validated by MS2 for both the sn-1 and sn-2 fatty acid substituents (Table 2). No prevs. post-differences in these lipids were monitored in the control group.

Other lipids

Supplementation was found not to alter any other glycerophospholipid not containing DHA. This included phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidyl glycerols, phosphatidyl inositols, choline plasmalogens, and ethanolamine plasmalogens.

Table 1: Effects of 60 days of a yeast-based supplement containing 1000 IU vitamin E, 2 mg selenium as selenized yeast, and 15 g algal docosahexaenoic acid (DHA) on the DHA/docosapentaenoic acid (DPA) ratio in stallion spermatozoa.

Table 2: Tandem mass spectral (MS2 ) analysis of diacyl and plasmenyl glycerophospholipids.

Currently there are limited lipidomics data for stallion spermatozoa in the literature [2-6]. Our data are the first to characterize the effects of a yeast-based supplement containing 15 g of algal DHA, 1000 IU vitamin E and 2 mg selenium, as selenized yeast, on individual ethanolamine, choline, and serine glycerophospholipids in equine sperm. Increases in DHAcontaining phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines were observed. Also, DHA-containing choline and ethanolamine plasmalogens were shown to be augmented by supplementation. Plasmalogens are essential ether lipids for sperm motility and direct binding of sperm with the zona pellucida of the oocyte [16,17]. As ether lipids, plasmalogens require peroxisomal enzymes for generating the ether linkage at sn-1 of the glycerol backbone [17]. In this regard, since spermatozoa are devoid of peroxisomes, it appears that peroxisomes in epithelial cells of the epididymis are probably the major source of these lipids. Epithelial cells are rich in peroxisomes in the rat [17], mouse [18], human [18], and horse [19].

While previous studies of equine spermatozoa have demonstrated the presence of C16:0 plasmalogens via quantitification of the dimethylacetyl derivative of hexadecanal [3] our studies were the first to analyze individual plasmalogens [6]. In the current study we further demonstrate that supplementation can augment the cellular levels of free DHA which in turn results in increased levels of a number of individual DHA-containing plasmalogens. A previous study [7] in stallions has demonstrated semen DHA levels increase with DHA supplementation; however, spermatozoa were not evaluated and the levels of DHA in the diet were 250 g/day, which were 16.6 times the levels in our study. DHA supplementation has been shown to improve sperm integrity, viability, and freezability in rabbits [8], chickens [9], and stallions [7].

Introduction of DHA into the glycerol backbone of glycerophospholipids occurs mainly via lipid remodeling, which involves coupled fatty acid deacylation with reacylation at sn-2 [20]. Our results demonstrate that in the case of phosphatidylcholines, deacylation coupled with DHA reacylation results in lowered levels of phosphatidylcholines containing 22:5 (PtdC 36:5, PtdC 40:5). However, in the case of choline plasmalogens, ethanolamine plasmalogens, phosphatidylethanolamines, and phosphatidylserines this was not the case. These data suggest that different enzyme isoforms may be involved in the deacylation-reacylation of sn-2 in phosphatidylcholines. The functional impact of augmented DHAcontaining phosphatidylcholines, in the presence of lowered 22:5-containing phosphatidylcholines remains to be evaluated.

In summary, our study provides the first detailed analysis of supplementation on the lipidome of spermatozoa from stallions. Supplementation was found to augment the levels of free DHA which in turn resulted in increased levels of DHA-containing diacyl and alkenyl-acyl glycerophospholipids essential for male fertility. Further studies are required to assess the effects of these changes on sperm function and fertility.

Our data demonstrate that supplementation can significantly increase the DHA to DPA ratio in the sperm of supplemented horses. In addition, we also demonstrate for the first time that these increases in free DHA act to augment the levels of 22:6-containing diacyl and alkenyl-acyl glycerophospholipids. While the supplement also contained antioxidants and yeast culture, the augmented lipid changes were specific to elevations in free DHA and the associated availability of DHA for lipid remodeling of glycerophospholipids.

This work was funded by DeBusk College of Osteopathic Medicine, Lincoln Memorial University, by the Albert G. Clay Endowment and by a grant from Alltech Inc. The authors thank Dr. Juan Gallardo, Dr. Shavahn Loux, Dr. Gabriel Davolli, Lauren Goedde, Morgan Pyles for technical assistance.

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