DIFFERENCES BETWEEN SHEEP AND RED DEER IN IN VITRO APPARENT AND TRUE DIGESTIBILITY OF COMMONLY USED RED DEER FEEDS

The nutritive value of red deer feeds is frequently determined by sheep despite the ultimate arbitrator of the nutritive value of any feed is the host animal. The objective of the trial was to determine the influence of rumen fluid donor (sheep vs red deer) on in vitro dry matter (DMD), neutral-detergent fibre (NDFD) and true digestibility (ivTD) of eleven substrata, naturally occurring in Slovenian forests (chestnut fruits, acorns of common and sessile oak, two fresh grasses) and those frequently used in supplemental red deer feeding (two grass hays and two grass silages, apple pomace and sugar beet roots). Only the fresh grass from Jelendol had greater (p < 0.05) DMD (646 vs 508 g/kg) when incubated in red deer inoculum. The NDFD and ivTD were always numerically greater when substrates were incubated in red deer inocula, however the NDFD and ivTD were significantly greater (p < 0.05) only when fresh grass from Jelendol (590 vs 343 g/kg and 801 vs 681 g/kg, respectively), grass silage from Kokra (541 vs 359 g/kg and 742 vs 639 g/kg, respectively) and apple pomace (428 vs 328 g/kg and 704 vs 653 g/kg, respectively) were incubated in the inoculum prepared from red deer rumen contents. These results indicate that rumen fluid from sheep can be used to predict in vitro digestibility in red deer and that these parameters can be used in the formulation of deer diets.


Differences between sheep and red deer in in vitro apparent and true digestibility of commonly used red deer feeds
The nutritive value of red deer feeds is frequently determined by sheep despite the ultimate arbitrator of the nutritive value of any feed is the host animal.The objective of the trial was to determine the influence of rumen fluid donor (sheep vs red deer) on in vitro dry matter (DMD), neutral-detergent fibre (NDFD) and true digestibility (ivTD) of eleven substrata, naturally occurring in Slovenian forests (chestnut fruits, acorns of common and sessile oak, two fresh grasses) and those frequently used in supplemental red deer feeding (two grass hays and two grass silages, apple pomace and sugar beet roots).Only the fresh grass from Jelendol had greater (p < 0.05) DMD (646 vs 508 g/kg) when incubated in red deer inoculum.The NDFD and ivTD were always numerically greater when substrates were incubated in red deer inocula, however the NDFD and ivTD were significantly greater (p < 0.05) only when fresh grass from Jelendol (590 vs 343 g/kg and 801 vs 681 g/kg, respectively), grass silage from Kokra (541 vs 359 g/kg and 742 vs 639 g/kg, respectively) and apple pomace (428 vs 328 g/kg and 704 vs 653 g/kg, respectively) were incubated in the inoculum prepared from red deer rumen contents.These results indicate that rumen fluid from sheep can be used to predict in vitro digestibility in red deer and that these parameters can be used in the formulation of deer diets.
Key words: animal nutrition; red deer; sheep; rumen; feed evaluation; in vitro digestibility; supplementary feeding

INTRODUCTION
The Alpine region of Slovenia is characterised by large areas of mixed coniferous and deciduous forests with small areas of pastures available for red deer.This area is also characterised by wet and cold winters.Under such environmental conditions, the herbage availability for grazing deer is characterised by a low quantity and a poor quality of herbage during late autumn and during the winter.Such limitations together with large herds of red deer in some areas create a need for supplementary feeding as a common strategy to improve animal condition, trophy quality and reduction of winter mortality and is also accepted as an efficient biological method to reduce the bark browsing caused by red deer (Rajský et al., 2008).
To obtain these goals it is essential to know the nutritive value of feed ingredients, especially for those feeds which are commonly used for the supplementary feeding of red deer and those who are commonly found in their environment.Currently, the nutritive value of feeds determined in sheep is used as guideline despite the fact that the ultimate arbitrator of the nutritive value of any feed is the host animal (Hervás et al., 2004;Ru et al., 2002).The use of domestic ruminants rather than deer greatly facilitates the in vitro digestion technique in that it eliminates problems associated with the deer as living rumen fluid donor or the necessity of sacrificing an animal (Palmer et al., 1976).
Most approaches to feed characterisation are intended to meet the needs of rationing systems and, because digestibility is the principal cause of variation of the metabolizable energy content, the energy value of feeds is commonly predicted from in vitro estimates of digestibility.With the increased use of in vitro techniques to evaluate ruminant feeds, it is of great importance to identify whether the species from which the rumen fluid inoculum is prepared has a significant influence on the obtained results.
This trial was carried out to determine the influence of rumen fluid donor (sheep vs red deer) on in vitro digestibility of several substrates naturally occurring in Slovenian forests and those frequently used in supplemental red deer feeding.

MATERIAL IN METHODS
Eleven feeds were used as substrates.In the areas of north Slovenia, Jelendol (community of Tržič) and Kokra (community of Preddvor), which are on average 767 and 940 m above the sea level, respectively, we collected samples of two fresh grasses, horse chestnut fruits (Aesculus hippocastanum) and acorns of common (Quercus robur) and sessile (Quercus petraea) oak.Other feeds (grass hays, grass silages, fresh sugar beets roots, apple pomace) were purchased and served as winter supplemental feeds for red deer.Samples of dried feeds were analysed for crude protein (CP), ash, ether extract (EE), crude fibre (CF) according to Neumann and Bassler (1986) and for neutral detergent fibre according to Goering and Van Soest (1970) using ANKOM 220 Fibre Analyser (Ankom Technology, Macedon, NY).Their chemical composition is given in Table 1.
Sheep rumen fluid was derived from two mature castrated Jezersko Solčavska × Romanovska rams (Ovis aries) weighing on average 70 kg and fitted with permanent rumen cannula.They received a daily ration containing approximately 1.0 kg average quality hay ad libitum and 0.5 kg of commercial compound feed (180 g/kg CP), supplemented with mineral and vitamin mix (25 g) once a day in the morning after rumen fluid collection.The diet composition was calculated according to the German metabolizable energy and utilizable protein requirements (nXP; DLG, 1997) to cover the energy and protein requirements for maintenance and to balance the energy-to-protein ratio in the rumen.Four red deer (Cervus elaphus) hinds aged 2 to 4 years were shot in areas of Jelendol and Kokra in September and October 2011 during the deer-stalking season according to the hunting action plan.Hinds did not show any signs of illness and were in body condition typical for the time in the season.Immediately after the hinds were shot the entire rumen was removed from the animal, placed in a warm polystyrene box and transported into the laboratory in no more than 45 minutes.Sheep rumen fluid and deer rumen were collected on different days because of equipment limitations.
In vitro incubations were carried out by using AN-KOM in vitro fermentation system Daisy II (ANKOM Technology, Macedon, NY, USA).Inoculum from each rumen fluid donor was prepared according to Menke and Steingass (1988).In the laboratory red deer rumen fluid was obtained by squeezing the rumen contents manually through four layers of cheese-cloth, while sheep rumen fluid was strained through four layers of cheesecloth.Obtained rumen fluids were diluted into the reduced buffer medium in the proportion 1-to-2 (v/v).Approximately 450 mg of ground air-dry sample milled through 1 mm screen was weighed into ANKOM F57 filter bag (ANKOM Technology, Macedon, NY, USA), heat sealed and placed in incubation jars.Each substrate was weighed into four bags, which were placed into 2 jars (2 bags/jar).Each jar contained 24 bags (2 bags/substrate + two blanks).Two litres of buffered rumen fluid dispensed under CO 2 were poured into each jar, which was then deposited into the rotating incubator at 39 °C for 24 h.At the completion of incubation, bags were rinsed thoroughly with cold tap water until the water was clear, dried and weighed to determine DM disappearance (DMD).Bags were then treated with boiling neutral detergent solution for 1 h using an ANKOM 220 fibre analyser (ANKOM Technology, Macedon, NY, USA), washed with distilled water, dried and weighed to determine both neutral detergent fibre disappearance (NDFD) and in vitro true DM digestibility (ivTD).Incubations and treatments with sheep inoculum were performed in two repetitions.The time from collection to completion of the inoculation was less than 2 hours as recommended by Schwartz and Nagy (1972).
One-way analysis of variance using the general linear model (GLM) procedure of the statistical package SAS/STAT version 9.4 (SAS Institute Inc., 2015) was used to compare the differences between animal species (sheep and red deer) for each substrate.Data are presented as least square means.Statistical significance was declared at p ≤ 0.05, whereas trends were discussed when 0.05 < p < 0.10.

RESULTS AND DISCUSSION
Chemical composition (Table 1) varies greatly between used substrates especially between forages (fresh grasses, grass hays and grass silages) and other used substrates (apple pomace, sugar beet roots, chestnut fruits and oak acorns).
The values of DMD, NDFD and ivTD (Table 2) show the expected variation depending on the substrate incubated, however, they show also some significant dif-ferences related to the species of the rumen inoculum donor (deer vs sheep).The considerably greater variation could exist among digestibility values from inoculums obtained from sacrificed red deer hinds, which could be a consequence of large variability of natural diets, who range from highly digestible fruits and seeds to poorly digestible browse.
In vitro DM disappearance was higher (p < 0.05) only for fresh grass from Jelendol when incubated in the inoculum obtained from deer.Crawford and Hankinson (1984) and Gordon et al. (2002) also did not find any differences in in vitro DMD using the inoculums prepared from white-tailed deer and cattle and white-tailed deer and cattle, respectively.However, Hervás et al. (2004) found that in vitro DMD were higher (p < 0.05) in sheep than in red deer of five out of eight studied forages.
The Tilley and Terry (1963) two-stage method for estimation of DM digestibility was used by Palmer et al. (1976) and Ru et al. (2002) to compare DM digestibility using inoculum from white-tailed deer, red deer and cattle.While Palmer et al. (1976) did not establish any differences in DM digestibility by using white-tailed deer and cattle inoculum, Ru et al. (2002) found that the substrates were generally better (p < 0.05) digested in inoculum obtained from red deer.They claimed that if the samples are incubated under the same conditions and the same diet was fed to animals before the rumen fluid was obtained then the differences in rumen digestibility are influenced only from differences in bacterial activity.However, in the present trial, the ivTD should represent total tract digestibility similar to Tilley and Terry (1963) method.The ivTD were always numerically higher when feeds were incubated in red deer inoculum; however, the differences were significant (p < 0.05) only for fresh grass from Jelendol, grass silage from Kokra and apple pomace.There was a trend (0.05 < p < 0.10) that grass silage from Jelendol and grass hay from Kokra had higher ivTD when incubated in inoculum obtained from red deer.On the contrary, Hervás et al. (2004) found that ivTD were higher (p < 0.05) in sheep than in red deer of five out of eight studied forages, while ivTD of barley and wheat grains were lower (p < 0.05) in sheep than in red deer.
There was no possibility to control the red deer diets.They consumed feeds naturally occurring in their environment, while sheep consumed the balanced diet prepared from conserved feeds and yet only a few substrates show differences (p < 0.05) in the DM digestibility.
It was assumed that the diet nutrient composition did not differ substantially between red deer and sheep, because red deer hinds inhabit sites with higher-quality forages than males (Barbosa and Bowyer, 2000), because Adamič (1990;cited by Jerina, 2007) noted that grasses comprise between 50 and 70 % of red deer diet in the vegetative season and because in the fall of 2011 (at the time of study) oaks, common beech and chestnut cropped heavily in the areas of Jelendol and Kokra (D. Veternik, personal communication, January 25 th , 2012).The later are rich sources of non-fibre carbohydrates (NFC, Table 1), mainly starch.Therefore, in this study, the red deer diet and sheep diet supplemented with compound feed had similar physical and chemical composition and thus should have also the comparable activity of rumen microorganisms in both studied species.These observations are supported also by the observation of Gordon et al. (2002) who reported that red deer rumen microor-ganisms adapted to high-quality diets digest these more efficiently than when low-quality diets are fed.
Similar to ivTD also the NDFD were always numerically higher when feeds were incubated in red deer inoculum (Table 2) and the differences were significant (p < 0.05) or tended to be significant (0.05 < p < 0.10) when comparing the two inoculums within the same feed.Hervás et al. (2004) also found that NDFD differed (p < 0.05) between inoculums for the same feed as did ivTD.The reason of identical statistical significancies is linked to the use of the neutral detergent solution in the determination of both, ivTD and NDFD.
The difficulty of generalizing the results on the digestion capacity of sheep and red deer imposed by the interaction between animal species and feed ingredients has been pointed out in the literature (Ru et al., 2002).Comparisons of digestion by sheep and deer have resulted in differences in favour of the deer or sheep and sometimes even in no differences depending on the feed studied.

CONCLUSIONS
Small differences in digestibility parameters, especially in in vitro DM disappearance (DMD) between sheep and red deer suggest that sheep inoculum could be used to predict DMDs of naturally occurring red deer feeds and feeds used frequently in their supplementary feeding.However, in vitro NDF disappearance (NDFD) and in vitro true DM digestibility (ivTD) were always higher when feeds were incubated in red deer inoculum, being significant (p < 0.05) or tended to be significant (0.05 < p < 0.10) for almost one-half of tested feeds.These differences are more prominent for forages than for high non-fibre carbohydrate (NFC) feeds, suggesting the higher fibrolytic activity of red deer rumen microorganisms, which could be a consequence of different microbial species inhabiting rumen of red deer (Henderson et al., 2015).Further research on this subject is needed in which the comparisons between deer and sheep are performed where sheep are fed with diets of different composition.

Table 2 :
In vitro dry matter (DMD; g/kg) and neutral-detergent fibre (NDFD; g/kg) disappearances and in vitro true dry matter digestibility (ivTD; g/kg) of different substrates incubated in the inoculum prepared from sheep and red deer rumen fluid